JXB Advance Access originally published online on April 11, 2008
Journal of Experimental Botany 2008 59(6):1279-1294; doi:10.1093/jxb/ern034
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Evidence for sugar signalling in the regulation of asparagine synthetase gene expressed in Phaseolus vulgaris roots and nodules
1Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av. Universidad 2001, Colonia Chamilpa, Cuernavaca, CP 62210, Morelos, México
2Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Colonia Chamilpa, Cuernavaca, CP 62210, Morelos, México
To whom correspondence should be addressed. E-mail: lara{at}ccg.unam.mx
Received 21 September 2007; Revised 22 January 2008 Accepted 23 January 2008
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Abstract |
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A cDNA clone, designated as PvNAS2, encoding asparagine amidotransferase (asparagine synthetase) was isolated from nodule tissue of common bean (Phaseolus vulgaris cv. Negro Jamapa). Southern blot analysis indicated that asparagine synthetase in bean is encoded by a small gene family. Northern analysis of RNAs from various plant organs demonstrated that PvNAS2 is highly expressed in roots, followed by nodules in which it is mainly induced during the early days of nitrogen fixation. Investigations with the PvNAS2 promoter gusA fusion revealed that the expression of PvNAS2 in roots is confined to vascular bundles and meristematic tissues, while in root nodules its expression is solely localized to vascular traces and outer cortical cells encompassing the central nitrogen-fixing zone, but never detected in either infected or non-infected cells located in the central region of the nodule. PvNAS2 is down-regulated when carbon availability is reduced in nodules, and the addition of sugars to the plants, mainly glucose, boosted its induction, leading to the increased asparagine production. In contrast to PvNAS2 expression and the concomitant asparagine synthesis, glucose supplement resulted in the reduction of ureide content in nodules. Studies with glucose analogues as well as hexokinase inhibitors suggested a role for hexokinase in the sugar-sensing mechanism that regulates PvNAS2 expression in roots. In light of the above results, it is proposed that, in bean, low carbon availability in nodules prompts the down-regulation of the asparagine synthetase enzyme and concomitantly asparagine production. Thereby a favourable environment is created for the efficient transfer of the amido group of glutamine for the synthesis of purines, and then ureide generation.
Key words: Asparagine and ureide synthesis, asparagine synthetase, nodules, Phaseolus vulgaris, sugar signalling
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Symbiotically reduced nitrogen in legume root nodules is assimilated via the incorporation of NH
into the amide position of glutamine in a reaction catalysed by glutamine synthetase. Subsequently, glutamate synthase (NADH-GOGAT) transfers the amido group of glutamine to 
-ketoglutarate producing two molecules of glutamate. In temperate and tropical legumes with indeterminate nodules, the symbiotically assimilated nitrogen is transported from nodules to the shoot in the form of amides (glutamine and asparagine), whereas some tropical legumes like cowpea (Vigna unguiculata), soybean (Glycine max), and bean (Phaseolus vulgaris), which produce determinate nodules, export ureides from nodules (allantoin and allantoic acid). Ureide production in nodules involves de novo synthesis of purine nucleotides in infected cells, followed by their degradation to give rise to allantoin and allantoic acid in uninfected cells (Atkins et al., 1982, 1991). Accordingly, as purine synthesis is initiated, the production of asparagine is reduced following the commencement of nitrogen fixation, because both purine and asparagine biosynthetic processes compete for the same amido group of the substrate glutamine for the synthesis of purine nucleotides and asparagine, respectively. As a result of this, ureides form the major nitrogen compounds that are exported, although asparagine continues to remain prominent among the amino acids found in the xylem sap of tropical legumes (Peoples et al., 1985; Leidi and Rodriguez-Navarro, 2000). This implies that nodule metabolism favours the transfer of the amido group of glutamine to the synthesis of purine nucleotides rather than for the synthesis of asparagine. However, the physiological basis that prompts ureide production rather than asparagine synthesis in nodules has not yet been deciphered.
The main route for asparagine biosynthesis in plants is via the glutamine-dependent asparagine synthetase (EC 6.3.5.4) enzyme (Ireland and Lea, 1999). Considerable progress has recently been made in understanding the molecular biology of plant asparagine synthetases. Asparagine synthetase cDNAs have been cloned from a number of legumes and non-legumes. In most plants examined, a single gene has been identified. However, some legumes contain two closely related genes that show similar regulatory patterns (Tsai and Coruzzi, 1990; Hughes et al., 1997; Osuna et al., 1999). In Arabidopsis (Lam et al., 1998) and sunflower (Herrera-Rodriguez et al., 2002), three asparagine synthetase genes have been identified.
Expression of asparagine synthetase genes is repressed by light and induced in the dark in a number of species such as soybean, common bean, alfalfa, maize, Arabidopsis, and sunflower (Lam et al., 1998; Moeller et al., 2003; Herrera-Rodriguez et al., 2004). However, in Lotus japonicus, asparagine synthetase expression seems unaffected by light (Waterhouse et al., 1996). Cis elements necessary for the repression by light have been identified in the asparagine synthetase promoter of pea (Ngai et al., 1997). It was also shown that the supply of sugars to dark–adapted Arabidopsis or glucose-starved maize root tips repressed the expression of asparagine synthetase genes (Lam et al., 1994; Chevalier et al., 1996). Light may control the expression of asparagine synthetase either directly as a physical signal, and/or because it affects the sugar content of the cell.
In plants, sugars produced by photosynthesis are used as an energy source and also act as physiological signals repressing or activating plant genes involved in many essential processes (Chen et al., 1994; Sheen, 1994; Mita et al., 1995; Reynolds and Smith, 1995). Even though the mechanism of sugar perception and signalling in plants remains unclear, hexokinase (HXK), sucrose and glucose transporters, and various sugar receptors have been proposed as components of the sugar-sensing machinery (Sheen et al., 1999; Smeekens, 2000; Rolland et al., 2002). Analysis of the asparagus asparagine synthetase gene promoter identifies evolutionarily conserved cis-regulatory elements that mediate sucrose-repression (Winichayakul et al., 2004), and it has been postulated that HXK might be involved in this regulation (Irving et al., 2000).
In this article, evidence is presented that the expression of the P. vulgaris (cv. Negro Jamapa) asparagine synthetase gene (PvNAS2) in roots and nodules is up-regulated by metabolizable sugars and that HXK has a role in the sugar-sensing mechanism that regulates the expression of PvNAS2. In conjunction with the sugar-dependent PvNAS2 regulation, the gene expression data demonstrating the lack of PvNAS2 expression in the central rhizobia-infected zone of the bean nodules suggest a mechanism that the suppression of asparagine synthesis in the nitrogen-fixing zone prompts purine and ureide production in the nodules of bean plants.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Plant growth
Bean (P. vulgaris 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. Germination was carried out on moist paper towels in sterile trays at 26 °C in the dark. Three days after germination, the seedlings were sown in vermiculite pots and inoculated with Rhizobium tropici CIAT899 and grown in a naturally lit greenhouse (May to August 2006). Plants were watered daily and, in addition, irrigated with N-free Summerfield solution (Summerfield et al., 1977) twice a week.
Isolation of PvNAS2 cDNA
A bean (P. vulgaris) nodule-specific cDNA library was screened for full-length asparagine synthetase clones by probing with a 657 bp fragment amplified by PCR with a forward primer (5'-GTT GAT CCT GCT TCT GGT G-3') and a reverse primer (5'-GCC CTT AAG GCC TAC ACA GA-3') derived from conserved sequence of legume asparagine synthetase genes. The PvNAS2 cDNAs from positive clones were subcloned into pBluescript SK (+) vector as EcoRI-XhoI fragments, and sequenced (Medigenomics, Munich, Germany).
Analysis of DNA and protein sequences
Homology searches against databases were performed using the BLAST program (Altschul et al., 1990). Multiple amino acid sequence alignments were made using the PileUp software package (Devereux et al., 1984).
Southern blot analysis
Genomic DNA was isolated from young bean leaves using the PUREGENE kit (Gentra Systems, Minneapolis, MN, USA) and Southern blotting was carried out according to Sambrook and Russell (2001). Genomic DNA (20 µg) was digested with XbaI, EcoRI, and HindIII, separated on a 0.8% agarose gel and transferred to a nylon membrane (Hybond N+, Amersham Pharmacia Biotech, Buckinghamshire, UK). Hybridization was carried out at 65 °C using a 32P-labelled 657 bp internal fragment from the PvNAS2 gene as a probe prepared by using a random primer DNA labelling kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The blots were prehybridized at 65 °C for 30 min in 300 mM phosphate buffer (pH 7.2) and 7% SDS. Hybridization was carried out in the same buffer at 65 °C for 24 h. The hybridized filters were 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 films.
RNA isolation, and northern and RT-PCR analyses
RNA was prepared from bean tissues using an RNA extraction kit (BIO-101, Vista, CA, USA).
For northern analysis, the total RNA loadings were adjusted to 20 µg per lane and separated by electrophoresis on 1.2% agarose gel containing formaldehyde, and blotted onto a nylon membrane (Hybond N+, Amersham Pharmacia Biotech). The membranes were hybridized, washed at a high stringency, and visualized as described in Silvente et al. (2003).
For reverse transcriptase-PCR (RT-PCR), cDNA was synthesized from 3 µg of total RNA, with oligo (dT)12–18 as the primer by using PowerScript preamplification system (Clontech, CA, USA), in a total volume of 20 µl. After reverse transcription, PCR was performed using PvAS2-derived primers (5'-CTA TAG AAT GAT CTT TGA GAG G-3' and 5'-GCA ATT AAA TAC GGT AAC CAC-3'; these primers were designed for the amplification of the region encompassing a part of the carboxy terminal plus the entire, unique 3' UTR), which were found to amplify PvNAS2 (an orthologue of PvAS2; Osuna et al., 1999) from the bean cv. Negro Jamapa as well. Each 25 µl reaction contained 1 µl of cDNA, 2.5 µl of 10x PCR buffer, 1.5 µl of 50 mM MgCl2, 1 µl of 2 mM deoxynucleotide triphosphates, 5 pmol of each primer, and 1 unit of recombinant Taq DNA polymerase (Altaenzymes, Canada). The amplification was carried out with an initial cycle of 1 min at 94 °C, followed by 30 cycles of 1 min at 94 °C, 30 s at 60 °C, and 1 min at 72 °C, and a single final cycle of 7 min at 72 °C. RT-PCR products were resolved on 1.2% (w/v) agarose gel in TRIS-acetate-EDTA buffer. Amplification of aquaporin transcript with the forward 5'-CGC CGC TGT TTG AGC CCT CG-3' and reverse 5'-TTG CGC ATC GTT TGG CAT CG-3' primers served as control for uniform PCR conditions.
Estimation of sugars
Glucose, fructose, and sucrose contents in nodules and roots were determined by enzymatic reactions coupled to the production of NADH, as described by Gordon et al. (1999).
Estimation of asparagine and ureide content of nodules
Nodules were ground in liquid nitrogen and extracted with 0.4 M potassium phosphate buffer (pH 7.4) at 300 mg ml–1. Cell-free extracts were prepared by centrifuging at 14 000 rpm in an Eppendorf centrifuge (Model 5415C; Brinkmann Instruments Inc., Welsbury, NY, USA) at 4 °C, and used for asparagine and ureide estimation.
Asparagine estimation in cell-free-extracts was essentially carried out using the method developed by Edman and Henschen (1975). Automated Edman derivatization of amino acids was performed with a Beckman Protein Sequencer (Model LF3000; Palo Alto, CA, USA) using Edman reagent consisting of phenylisothiocyanate (PITC). Subsequent to derivatization, the cleavage step required for amino acid analysis in peptides was omitted, as the aim was to analyse only the free amino acids (specifically asparagine) present in the cell-free extracts. Phenylthiohydantoin (PTH)-derivatized amino acids of the cell-free extracts were separated by an HPLC system equipped with an UV Detector (Model 168; Beckman) using a Micro PTH C18 column (Beckman). Elution was performed with a gradient from 7.5% to 52% acetonitrile in combination with tetrahydrofuran for 14.2 min at a flow rate of 0.2 ml min–1. The derivatized amino acid peaks were monitored at 268 nm. Authentic asparagine dissolved at a concentration of 4 nmol in phosphate buffer served as a standard in HPLC analysis.
Concentration of ureides present in nodule cell-free extracts was measured using the colorimetric detection method of Vogels and Van der Drift (1970). Allantoic acid dissolved at a concentration of 10 mM in water served as a standard for ureide estimation.
PvNAS2 promoter isolation and construction of plant transformation vector
Genomic DNA prepared from young bean leaves was utilized for generating ‘GenomeWalker Libraries’ using a Universal GenomeWalker kit (Clontech) according to the manufacturer's instructions. The 5' flanking genomic region of PvNAS2 was amplified by using two rounds of genome walking by long-distance nested PCR using bean ‘GenomeWalker Libraries’ and Advantage Genomic Polymerase Mix (Clontech). Nested adaptor primers and gene specific primers used for genome walking of the 5' flanking sequence of PvNAS2 are given in Supplementary Table S1 at JXB online. The long-distance PCR cycles used were as follows. Primary PCR amplification was carried out with five initial cycles of 28 s at 94 °C and 4 min at 72 °C, followed by 20 cycles of 28 s at 94 °C and 4 min at 67 °C, and a single final cycle of 7 min at 67 °C. Secondary PCR was carried out with an initial cycle of 1 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 1 min at 64 °C, and 2 min at 72 °C, and a single final cycle of 7 min at 72 °C. Isolated 5' flanking regions were cloned into the pGEM vector (Promega, USA) and sequenced at Medigenomics (Munich, Germany), and the transcription start site was determined using the Neural Network Promoter Prediction Program (http://www.bdgp.org/seq_tools/promoter.html) in conjunction with the untranslated 5' sequence of PvNAS2 cDNA. The promoter sequence was further analysed using the plant cis-acting regulatory DNA elements (PLACE) database (Higo et al., 1999; http://www.dna.affrc.go.jp/PLACE/) as well as the plant cis-acting regulatory element database (Lescot et al., 2002; http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
The binary vector pBI-PrPvNAS2-GUS containing the P. vulgaris asparagine synthetase promoter transcriptionally coupled to the gusA coding sequence and the 3' Nos terminator sequence was constructed as follows. An 842 bp PrPvNAS2 upstream of the ATG codon of the asparagine synthetase gene was PCR amplified using the PvAS For1 and PvAS Rev1 primers (see Supplementary Table S1 at JXB online) and Advantage 2 Polymerase Mix (Clontech). The amplification was carried out using seven initial cycles of 30 s at 94 °C and 3 min at 72 °C, followed by 28 cycles of 30 s at 94 °C and 3 min at 68 °C, and a single final cycle of 7 min at 68 °C. The PCR product was sequenced, digested with SalI and XmaI (these restriction sites were embedded in PvAS For1 and PvAS Rev1 primers; see Supplementary Table S1 at JXB online) and cloned upstream of the gusA coding sequence in a similarly digested pBI101.1 binary vector (Clontech) yielding pBI-PrPvNAS2-GUS.
Development of Agrobacterium rhizogenes strains carrying binary vectors and inoculum preparation
Binary vectors used for bean transformation, pBI101.1 (control vector) or pBI-PrPvNAS2-GUS, were introduced into Agrobacterium rhizogenes K599 by electroporation (Nagel et al., 1990). A. rhizogenes strains harbouring the transformation vectors were grown at 30 °C in Luria-Bertani (LB) plates supplemented with 50 µg ml–1 kanamycin. The bacterial cells from a culture grown overnight from a single plate were harvested and resuspended in 5 ml sterile distilled water, and used for infecting bean seedlings for the induction of hairy root formation (see below).
Hairy root transformation, plant culture, and rhizobial inoculations
Bean seeds obtained from greenhouse-grown plants were initially washed with 10% Extran MA 03 (Merck KGaA, Germany) detergent solution for 1 min followed by two washes with sterile distilled water, and surface-sterilized by treating with 70% (v/v) ethanol for 1 min followed by a treatment with 10% commercial bleach (Cloralex; Alen S.A. de C.V., Mexico) for 10 min. Seeds were then washed extensively with sterile distilled water, and left overnight in fresh sterile distilled water to soak before germination. Seeds were germinated in pots containing sterile vermiculite, incubated in the plant growth room (maintained at a 14/10 h light/dark cycle and a temperature of 25 °C) and irrigated with Summerfield nutrient solution (Summerfield et al., 1977) supplemented with 5 mM KNO3. Five to six days after planting, plantlets with newly unfolded cotyledons were infected by injecting A. rhizogenes strains carrying the binary vectors pBI101.1 (control vector) or pBI-PrPvNAS2-GUS at the cotyledonary nodes with a syringe as described by Estrada-Navarrete et al. (2006). Ten to twelve days after infection, plantlets exhibiting profuse hairy root formation at the site of infection were selected, primary root removed by cutting approximately 1 cm below the cotyledonary node and replanted in fresh pots containing sterile vermiculite. Immediately after transferring to new pots, each of the A. rhizogenes-transformed composite plants were inoculated with 1–2 ml R. tropici CIAT899 culture (rhizobial inoculum was prepared by growing CIAT899 strain to a density of 106 cells ml–1 in peptone-yeast extract (PY) medium supplemented with nalidixic acid, 20 µg ml–1), and irrigated with Summerfield nutrient solution (Summerfield et al., 1977) containing a highly reduced level (0.385 mM) of KNO3. Subsequently, the plants were covered with polyethylene bags to maintain humid conditions, and returned to the plant growth chamber maintained at a 14/10 h light/dark cycle and a temperature of 25 °C. After 4–5 d of incubation, the plastic bags were perforated to facilitate the gradual acclimation of transformed plants to the environment for a few days, before they were transferred to the naturally lit greenhouse maintained at 25 °C. The plants were harvested 3–4 weeks after rhizobial inoculation, and roots and nodules were analysed for GUS activity.
Histochemical localization of GUS activity and microscopical methods
GUS staining was essentially the same as that described by Reddy et al. (1998). Briefly, hairy roots with nodules were excised from the transformed plants, washed twice with 0.1 M potassium phosphate buffer, pH 7.0 and immersed in the GUS substrate solution containing 1 mM X-gluc (5-bromo-4-chloro-3-indolyl glucuronide, sodium salt; Biosynth AG, Switzerland), 10 mM EDTA, 0.1% Triton X-100, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 100 mM potassium phosphate buffer, pH 7.0. Incubation was performed in the dark at 30° C for 12–24 h. After rinsing in phosphate buffer, stained tissues were fixed for more than 4 h in a solution containing 3.7% formaldehyde, 5% acetic acid, and 50% ethanol, examined as whole specimens, and photographed under a stereomicroscope (Carl Zeiss Stemi 2000-C, Germany). For visualizing tissue-specific expression of the PvNAS2 promoter, the GUS-stained roots and nodules were cross-linked with 100 µM m-maleimidobenzoyl-N-hydroxysuccinimide-ester (MBS), fixed with 4% paraformaldehyde, processed through a dehydration series, and hand-sectioned (approximately 50–100 µm) according to Estrada-Navarrete et al. (2006). For obtaining 3 µm thick sections, the cross-linked GUS-stained materials processed as described above were embedded in methacrylate resin (Estrada-Navarrete et al., 2006) and sectioned using an ultramicrotome (Leica, Bensheim, Germany). The tissue sections were mounted on a slide with Citiflour (Ted Pella, Inc., Redding, CA, USA) and observed under a bright-field microscope (Zeiss Axioscop, Germany).
Sequence data from this article have been deposited in the Genbank under the accession numbers PvNAS2 promoter, EF620933 [GenBank] ; PvNAS2 cDNA, EF623893.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Cloning and characterization of PvNAS2 cDNA
A full-length asparagine synthetase cDNA clone, designated as PvNAS2, was isolated from a 15-d-old nodule cDNA library of the tropical legume P. vulgaris cv. Negro Jamapa (Silvente et al., 2003), using a 657 bp PCR product (see Materials and methods) derived from the conserved domain of the asparagine synthetase gene as a probe. Sequencing revealed that the PvNAS2 cDNA clone is 2032 bp long, and contained an open reading frame of 1752 bp, which corresponded to encoding a predicted protein of 584 amino acid-long, with a calculated molecular weight (Mr) of 65 800 (see Supplementary Fig. S1 at JXB online). At the 3' UTR, a polyadenylation signal (AATAAGA) sequence was detected 16 bp upstream of the poly(A) tail. PvNAS2 shared 85–99% identity and 94–99% similarity at the amino acid level with other reported asparagine synthetases (AS2 species) from various leguminous plants (Fig. 1). PvNAS2 lacks the transit peptide sequence for targeting into an organelle, suggesting a cytoplasmic location for this protein product. Earlier, Osuna et al. (1999) reported the isolation of an asparagine synthetase cDNA clone designated as PvAS2 from P. vulgaris cv. Great Northern. A comparison of PvNAS2 and PvAS2 cDNAs revealed that they are homologous, excepting that the isolated PvNAS2 cDNA clone is found to be longer by 53 bp at the 5' end, but lacked a 56 bp fragment at the 3' end between the polyadenylation signal and the poly(A) tail. In addition to these differences, eight nucleotide substitutions were detected in the coding region of PvNAS2 (see Supplementary Fig. S1 at JXB online). However, only two nucleotide substitutions from G to A at position 674 and from G to C at position 1097 resulted in two amino acid changes in the conserved domain, respectively leading to Asp and Thr residues in PvNAS2 instead of a Gly and Arg as in PvAS2. In contrast, the six other nucleotide observed substitutions did not lead to modification in the encoded amino acids.
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Genomic organization
The genetic complexity of PvNAS2 was investigated by gel-blot analysis of total genomic DNA. The PvNAS2 cDNA contained two XbaI sites, but no EcoRI, and HindIII sites. When bean genomic DNA digested with XbaI, EcoRI or HindIII was subjected to Southern blotting with the 657 bp internal fragment of the PvNAS2 cDNA as a probe at high stringency, two to four hybridizing bands per lane were observed, with one band being more intense than the other remaining bands (Fig. 2). These results are consistent with the idea that asparagine synthetase in bean is encoded by a small gene family (Osuna et al., 1999).
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It may be worthwhile to mention here that seven asparagine synthetase ESTs have been identified from root, nodule, and pod cDNA libraries prepared from the bean cv. Negro Jamapa. Sequence data showed that, of these seven ESTs, four (GenBank accession nos. CV541315, CV542782, CV543824, and CV544181) are identical to the PvNAS2 clone (and thus belong to the same contig corresponding to a unigene), while each of the remaining three ESTs (CV543090, CV539591, and CV535719) represented distinct and independent AS cDNA species. Based on these results it may be reasonable to suggest that asparagine synthetase in cv. Negro Jamapa is encoded by four different genes, including PvNAS2, which is orthologous to PvAS2 from the bean cv. Great Northern reported by Osuna et al. (1999), but exhibited a distinct feature from PvAS2 with respect to its regulation in response to sugars (see below).
The 3' UTR of PvNAS2 is post-transcriptionally processed in the bean cv. Negro Jamapa
Comparative analysis of the sequences of the AS ESTs in conjunction with the PvNAS2 clone indicated that in Negro Jamapa four distinct species of AS transcripts are expressed (see above). In addition to this, sequence analysis further revealed that the PvNAS2 ESTs (CV541315, CV542782, and CV543824) carried 3' UTRs of varied lengths (see Supplementary Table S2 and Fig. S2 at JXB online), indicating the occurrence of heterogeneity in 3' UTR sequences among PvNAS2 transcripts. At this juncture, it is important to mention here that we were also able to amplify PvNAS2 from cDNA prepared from the nodule tissue of the bean cv. Negro Jamapa using the 3'-specific primers generated based on the carboxy terminal plus the entire 3' UTR sequence of the orthologue PvAS2 from the bean cv. Great Northern (Fig. 5); thus providing clear evidence for the occurrence of the PvNAS2 transcript with an extended 3' UTR in the cv. Negro Jamapa, similar to that present in PvAS2 from the bean cv. Great Northern (Osuna et al., 1999). Based on these findings it may be suggested that the PvNAS2 transcript is subjected to post-transcriptional processing in cv. Negro Jamapa. Future studies can only unravel the significance of varied extents of 3' UTR in PvNAS2. In an analogous situation in rice, it has been observed that the transcripts of AS (a homologue of PvNAS2/PvAS2) derived from callus tissue also displayed heterogeneity in the 3' UTR region (Nakano et al., 2000) similar to PvNAS2.
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Expression of PvNAS2 in various plant organs
As part of our efforts to explore the role of asparagine synthetase in the nitrogen metabolism of bean plant, we initially set out to analyse the expression of PvNAS2 in various plant organs. Northern blot analysis of RNA from different plant parts showed the highest level of PvNAS2 transcript in roots and the lowest level in the aerial parts of the plant (Fig. 3A). In addition to roots, PvNAS2 is also abundantly expressed in nodules. Expression analysis during the various stages of nodule development showed that PvNAS2 mRNA transcript level is highest at the early stages of nodule formation until about day 18, and thereafter the transcript level declined and remained low until the end of nitrogen fixation activity (day 30; Fig. 3B). In bean, since the highest nitrogen-fixing activity occurs during the third week of nodule development (Ortega et al., 1992), the observed maximal level of expression of PvNAS2 in nodules during this period seems to suggest that the highest activity of PvNAS2 is restricted to the initial phase of nitrogen fixation, and the gradual decline in the expressional levels of PvNAS2 during the subsequent stages of nodule maturation possibly lead to reduced asparagine synthesis, as expected for a ureide transporter legume such as bean (see below).
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Expression of PvNAS2 is regulated by carbon availability in nodules and roots
Since carbon accessibility is a key limiting factor for symbiotic nitrogen fixation and nitrogen assimilation (Ortega et al., 1992), and because the sugar content decreases during nodule maturation (Silvente et al., 2003), it was therefore tested whether the down-regulation of PvNAS2 during nodule maturation is influenced by the availability of sugars. To test this hypothesis, PvNAS2 mRNA levels in the nodules of 19-d-old plants grown in a normal day/night cycle (control; treatment C in Fig. 3C) were compared with those in the nodules from plants adapted in darkness for 48 h, irrigated with (treatments S and G in Fig. 3C) or without (treatment D in Fig. 3C) 3% sucrose or glucose solution. In addition, the mRNA levels were also tested in the nodules of the 48 h dark-adapted plants that were subsequently transferred to a single day/night cycle after the dark treatment (treatment D/L in Fig. 3C) in order to assess if light can mimic the effect of sugar on nodule PvNAS2 mRNA expression. In all treatments, nodules were always collected at the end of the dark period in order to maintain the uniformity in sampling time and to avoid the induction of light-triggered changes in carbon content at the termination of each treatment, which might influence PvNAS2 expression. Northern blot analysis of mRNAs derived from the nodules obtained from variously treated plants revealed that the PvNAS2 transcript level in the nodules from 48 h dark-adapted plants remained almost at a similar level as that in control plants that were subjected to a normal light/dark cycle (compare the treatments C and D in Fig. 3C). However, PvNAS2 transcript levels were found to decrease when the dark incubation period was extended beyond 48 h (data not shown). On the other hand, in the plants shifted from 48 h dark to a light/dark cycle for 24 h in the absence of sugars (treatment D/L in Fig. 3C), nodule PvNAS2 mRNA levels slightly increased as compared to the control plants (treatment C in Fig. 3C). However, supplementation of plants with either sucrose or glucose led to the accumulation of high levels of PvNAS2 transcript (treatments S and G in Fig. 3C). These results clearly indicated that PvNAS2 expression is up-regulated in nodules with the increase in the availability of sugars.
The inductive effect of sucrose and glucose on PvNAS2 expression in nodules prompted us to test the effects of different carbon sources on the expression of the PvNAS2 gene in roots. For this purpose, the roots excised from 3-d-old dark-grown seedlings were incubated in solutions containing various concentrations of sugars for 24 h in the dark, and assessed for PvNAS2 mRNA induction by northern analysis. Roots freshly derived from cotyledon-bearing intact seedlings (treatment ‘0’ in Fig. 4A), and the detached roots incubated in sterile distilled water (treatment ‘C’ in Fig. 4A) served as controls. Within 24 h of incubation in water, as compared to the roots newly obtained from the intact seedlings, the roots that were severed from the cotyledons showed a dramatic decrease in PvNAS2 transcript levels (compare ‘0’ and ‘C’ treatments in Fig. 4A), implicating the essential role for cotyledons in supporting (probably by supplying sugars) PvNAS2 expression. On the other hand, the detached roots when supplied with sucrose, glucose, and fructose showed an enhanced induction of PvNAS2 (Fig. 4A), supporting the idea that the continuous supply of sugars is essential for sustaining the expression of PvNAS2 in roots. Maximal induction of PvNAS2 in roots was achieved with the supplementation of glucose compared with sucrose in a range from 0.5–4% concentration, the glucose response being dosage-dependent (Fig. 4C). Feeding roots with mannitol, a non-metabolizable carbon source, did not promote the expression of PvNAS2, as the level of induction was found to be similar to that in water-treated control roots (compare ‘M’ and ‘C’ treatments in Fig. 4A). These results indicated that the induction of PvNAS2 expression by sucrose, glucose, and fructose was not due to an osmotic effect (Fig. 4A).
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Quantitative analysis of the sugars revealed that the internal reserves of metabolizable sugars are indeed enhanced in the nodules/roots when supplied with exogenous sucrose, fructose, or glucose (Figs 3D, 4B), and the evident rise in PvNAS2 transcript levels, in response to sucrose and glucose/fructose supplementation, in nodules/roots (Figs 3C, 4A, C) was found to be consistent with the increase in the accumulation of metabolizable sugars in these organs (Figs 3D, 4B). However, the observed difference in sucrose-, glucose-, and fructose-mediated PvNAS2 induction could possibly be due to the role of these sugars in the activation of different signal pathways, as has been proposed by Smeekens (2000).
Participation of hexokinase/sugar-sensing mechanism in the regulation of PvNAS2 expression
The flux of hexose sugar substrates through the HXK-catalysed reaction has been proposed as a primary signalling event in the sugar-sensing mechanism in many organisms, including plants (Gibson, 2000; Koch et al., 2000; Smeekens, 2000). A number of sugar analogues has been widely used to assess the involvement of HXK as a sugar sensor. In order to determine whether an HXK-mediated sugar-sensing mechanism is involved in the regulation of PvNAS2 expression in bean, the effects of various sugar analogues on the sugar-induced expression of PvNAS2 in roots obtained from 3-d old seedlings as described above were evaluated. Northern blot analysis of RNA revealed that the metabolizable glucose analogue mannose, which can be phosphorylated by HXK (Herold and Lewis, 1977; Jang and Sheen, 1994), activated the expression of the PvNAS2 gene in roots (Fig. 4D). On the other hand, 3-O-methyl glucose, which is readily taken up by cells but is a poor substrate of HXKs (Graham et al., 1994), did not trigger induction of PvNAS2 expression. Furthermore, addition of glucosamine, an HXK inhibitor (Hofmann and Roitsch, 2000), to the roots inhibited the induction of sucrose-, glucose- or fructose-mediated PvNAS2 expression (Fig. 4D), thereby confirming the role for HXK activity in the regulation of PvNAS2 gene expression. These results together with those described in the earlier section suggest that the observed glucose induction of PvNAS2 expression operates via an HXK-mediated signalling pathway.
Verification of sugar-mediated enhancement in PvNAS2 transcript levels using RT-PCR with unique 3' primers
Even though the northern analyses in the present investigation were performed utilizing a probe derived from the coding region specific for PvNAS2, an uncertainty can still arise that the probe might have hybridized with extraneous mRNA products transcribed from other members of the AS gene family. To clarify this crucial point, a subset of experiments employing RT-PCR with gene-specific primers derived from the carboxy terminal and the unique, extended 3' UTR region of PvAS2 (see Materials and methods section) was performed using the nodule cDNA isolated from the plants subjected to various sugar treatments. RT-PCR results obtained with the 3' primers showed that the expression of PvNAS2 is up-regulated in the nodules supplemented with exogenous sucrose and glucose (Fig. 5). These results are in full agreement with the findings obtained with the northern analysis (Fig. 3C), thus confirming that the bands visualized on the northern blots correspond to that of the expression levels of PvNAS2 transcripts.
Glucose availability promotes asparagine synthesis while its deprivation favours ureide production in bean nodules
As increased sugar availability promoted PvNAS2 expression (Fig. 3C), it was verified whether such an up-regulation of PvNAS2 leads to a concomitant increase in asparagine production in nodules. For this study, nodules harvested from the bean plants (19-d-old) that were dark-adapted for 48 h and challenged with 3% glucose-containing medium during the dark phase were assessed for asparagine content. Nodules derived from 19-d-old plants grown under normal conditions served as controls. The data obtained with this study demonstrated that the feeding of plants with glucose indeed promoted asparagine production in nodules as compared to the control plants that were not supplied with glucose (Fig. 6).
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) or decrease (
) of asparagine or ureide, respectively, in the presence of glucose relative to their corresponding controls.Since both asparagine and ureide syntheses compete for the amido group of glutamine, there was a need to assess whether the enhancement of asparagine synthesis, with the addition of glucose, brings about the concomitant reduction in the production of ureides in nodules. Estimation of ureide content of the nodules was performed with the same plant samples (derived from the plants irrigated with or without glucose) that were used for asparagine analysis (see above). As is shown in Fig. 6, when glucose was added to the dark-adapted nodulated plants, the ureide production declined by about 30% in the nodules compared with the nodules derived from the plants that were not treated with glucose (control).
Together, the above results demonstrated that under glucose-sufficient conditions asparagine production in nodules is enhanced, while at the same time ureide content is reduced. In contrast, under glucose-deficient conditions, ureide production is increased as the asparagine synthesis is decreased. At this juncture it should be emphasized here that the fall in ureide production in the glucose-supplemented nodules is not due to the repressive effect of glucose on nitrogen fixation, as it was shown that the nitrogen-fixing activity in nodules is enhanced when the bean plants were augmented with the external sources of sugars (see Supplementary Fig. S3 at JXB online). Thus it is likely that the down-regulation of PvNAS2 due to sugar starvation during the later stages of nodule maturation (Fig. 3B) allows enhanced utilization of the amide group of glutamine for the purine synthesis through the glutamine dependent PRPP-amidotransferase, instead of consumption of the amide group for the synthesis of asparagine through asparagine synthetase. In subsequent stages, degradation of purine nucleotides leads to ureide production in nodules.
Isolation and characterization of PvNAS2 promoter
With a plan to study the pattern of PvNAS2 gene expression during root and nodule development, the aim initially was to isolate the promoter region of the PvNAS2 gene from bean genomic DNA by using a genome walking strategy (see Materials and methods). The cloned PvNAS2 promoter was determined to be 842 bp long (Fig. 7). Determination of the transcriptional start site in the promoter region using the Neural Network Promoter Prediction Program (see Materials and methods) delineated a 98 bp long 5' untranslated region in PvNAS2 cDNA. A TATA box (TATAAAT) was recognized at positions –122 to –128 upstream of the translational initiation site (+1). A putative CAAT box motif was found to be situated at –326 to –329. Thus the proximal region of the PvNAS2 promoter comprises the elements shown to be necessary for an accurate initiation of basal transcription in promoters of other plants. A search for locating cis-acting elements in the PrPvNAS2, using PLACE and PlantCARE databases, revealed several putative regulatory motifs that have been shown to be involved in the regulation of tissue-specific gene expression as well as the expression in response to a variety of biotic and abiotic factors. In the promoter of PvNAS2, several sugar-responsive cis-acting elements were found such as TGGACGG for sucrose-inducible expression (Morikami et al., 2005), the G-box element CACGTG (Urwin and Jenkins, 1997), CGACG element (Hwang et al., 1998), and the pyrimidine box motif CCTTTT (Morita et al., 1998) that are responsible for sugar repression, and the W-box element TGACT (Sun et al., 2003) that participates in sugar signalling (Fig. 7). In addition, in the PrPvNAS2, cis-acting elements were also found that were shown to be consensus sequences (CTCTT, AAAGAT, and AATAA) for nodule-specific expression (Sandal et al., 1987; Stougaard et al., 1990; Vieweg et al., 2004; Fehlberg et al., 2005), the regulatory motifs GCCACT box (CAT box) and CCGTCC box related to meristem-specific expression (Meshi et al., 2000), and CAGAAGATA motif required for phloem-specific gene expression (Yin et al., 1997) (Fig. 7).
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TATA Box,
transcription start site and various motifs for sugar responsive and organ-/tissue-specific expression. Sugar responsive elements:
TGGACGG cis-acting regulatory element involved in the sucrose-inducible expression of the sporamin gene promoter from sweet potato (
the G-Box element CACGTG: a sucrose repression element in the P. vulgaris rbcS2 gene promoter (
CGACG element found in the GC-rich regions of the rice Amy3D and Amy3E amylase genes, and functions as a coupling element for the G-box element during sugar starvation (
the W-box element TGACT: a novel WRKY transcription factor, SUSIBA2, binds to W-box and participates in sugar signalling in barley by binding to the sugar-responsive elements of the iso1 promoter (
the pyrimidine-box CCTTTT found in rice 
AATAA,
CTCTT, and 
AAAGAT: consensus sequences for nodule-specific expression (
CAT (GCCACT) box, and
CCGTCC box: A. thaliana cis-acting regulatory elements related to meristem-specific expression (
CAGAAGATA motif is required for phloem-specific gene expression in RTBV promoter in rice (The PvNAS2 promoter-GUS fusion is expressed in meristematic tissues and vascular traces, but not in infected and uninfected cells located in the central nitrogen-fixing zone of nodules
To determine the pattern of PvNAS2 gene expression in plants grown under symbiotic conditions, the PvNAS2 promoter fused to the gusA (uidA) reporter gene (Fig. 8A) was used and composite bean plants with transgenic hairy roots were generated through A. rhizogenes-mediated transformation (see Materials and methods). GUS staining of the roots from the symbiotically grown plants (see Materials and methods) revealed that the PvNAS2 promoter is highly active in roots and nodules, although the intensity of expression varied depending on the developmental stage (Fig. 8B). In meristematically active root zones, intense GUS staining was observed in the meristematic cells located at the root tip and in the vascular tissues, and less intensely in the cortical cells (Fig. 8B, a). On the other hand, in the fully matured region, GUS expression was predominantly confined to vascular tissues (Fig. 8B, c). During the processes of lateral root differentiation, PrPvNAS2-gusA expression was initially intense in the primordium and the nascent cortical cells surrounding the emerging lateral root (Fig. 8B, b), while in the following stages of the development, GUS expression was mainly limited to vascular bundles (not shown). In the matured regions of the main and lateral roots, PrPvNAS2-gusA expression in the vascular bundles was restricted to the endodermis, pericycle, and phloem parenchyma (Fig. 8B, d, arrows). In addition, histochemical staining of the roots revealed strong GUS expression in the developing nodule primordia (Fig. 8B, e, f) and mature nodules (Fig. 8B, g). In fully developed nodules GUS activity was mainly restricted to the outer cortical cell zone (Fig. 8B, h) and vascular tissues (Fig. 8B, h, arrows), particularly the pericycle, endodermis, and phloem parenchyma (Fig. 8B, i, j). PrPvNAS2-gusA expression was never observed in infected as well as uninfected cells located in the central zone of mature nodules (Fig. 8B, h). Lack of PvNAS2 expression in the infected and uninfected cells in the nitrogen-fixing zone indicates that these cells may not be the sites of asparagine synthesis in mature bean nodules.
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Expression of PvNAS2 promoter is induced by glucose and sucrose
In order to determine whether the addition of sugars such as glucose or sucrose modulates the activity of the PvNAS2 promoter, the young composite bean plantlets with hairy roots harbouring PrPvNAS2-gusA were treated with 3% glucose or sucrose for 3 d and analysed for GUS expression. Supplementation of plants with either glucose or sucrose stimulated GUS expression in transgenic roots, thus clearly indicating that the expression of the PvNAS2 promoter is induced with the increase in the availability of sugars (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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A cDNA clone coding for glutamine-dependent asparagine synthetase has been isolated from a bean nodule cDNA library. Northern analysis of PvNAS2 expression showed that, although induced in all tissues tested, it is preferentially expressed in roots, and to a lower extent in nodules (Fig. 3A). Analysis of PvNAS2 during nodule development showed a high induction of PvNAS2 mRNA between 9 dpi and 18 dpi, but exhibited a significant decline thereafter (Fig. 3B). These data suggests a role for PvNAS2 in nodule metabolism particularly at 9–18 dpi.
In plants, sugars have conventionally been viewed as resources for metabolic intermediates as well as structural or storage components, but recent experiments have provided evidence that sugars are also somehow involved in signalling pathways (Smeekens, 2000; Gibson, 2005). Sugar analogues are often used to obtain information about sugar-response pathways in various organisms including plants (Gibson, 2000). In the present study, it was found that the sugars, mainly glucose, induced the expression of PvNAS2 (Figs 3C, 4A, C). Investigations with sugar analogues revealed that mannose, which is a substrate for phosphorylation by HXK (Herold and Lewis, 1977; Jang and Sheen, 1994) but is poorly further metabolized, caused the induction of PvNAS2, while 3-O-methyl glucose, which can enter the cell but is a poor substrate for HXK (Graham et al., 1994), could not trigger the expression of PvNAS2 (Fig. 4D). Furthermore, glucosamine an inhibitor of HXK activity (Hofmann and Roitsch, 2000) prevented sugar-mediated induction of PvNAS2. Based on these results, it is proposed that the observed glucose induction of PvNAS2 expression in bean operates via a HXK-mediated signalling pathway. In contrast, the AS1 gene in Arabidopsis is found to be repressed by sugars through an HXK independent pathway (Xiao et al., 2000).
Results obtained with sugar-induced expression of PvNAS2, however, contrasted with those reported in etiolated and light-grown seedlings of A. thaliana (Thum et al., 2003), suspension cell culture of asparagus (Davies et al., 1996), and in roots of maize (Chevalier et al., 1996) and bean (PvAS2; Osuna et al., 1999), where the expression of asparagine synthetase is inhibited by sugars. The contrasting effect of glucose/sucrose on the expression of PvNAS2 (induction) and PvAS2 (inhibition) is intriguing. The observed contrasting effect of metabolizable sugars on the induction of PvNAS2 in the cv. Negro Jamapa (present study) and the inhibition of PvAS2 in the cv. Great Northern (Osuna et al., 1999) may be due to the cultivar differences, as well as the disparity in the age of nodules used in the present study (2–3-week-old) and those employed by Osuna et al. (5-week-old). In this context it is worthwhile to mention here that, in the cells of the cv. Negro Jamapa, the transcripts of PvNAS2 (unlike PvAS2) occur with a wide-range of 3' UTR lengths (see Supplementary Table S2 and Fig. S2 at JXB online). It has been reported in maize that a sequence of 150 bp (3' UTR) in Incw1-L between the polyadenylation signal and the poly(A) tail is a main factor responsible for sugar regulation of the two Incw1 isoforms (Cheng et al., 1999). Future studies can only resolve whether the heterogeneity in the length of 3' UTR region in PvNAS2 has any role to play in the differential regulation of PvNAS2 in response to sugars.
Interaction between cis-regulatory elements present in a promoter region and transacting factors is an essential prerequisite for activating gene expression. Specific cis-regulatory elements in the promoter are probably shared by the genes that are responsive to similar cellular cues/factors. In silico analysis against the plant regulatory elements databases revealed the presence of cis sequences responsible for sugar regulation in the promoter of PvNAS2 (Fig. 7). These cis sequences included conserved sugar responsive elements which are found in the promoter sequences of some sugar-regulated genes, such as sporamine and -amylase genes in sweet potato and rice (Ishiguro and Nakamura, 1992; Hwang et al., 1998; Maeo et al., 2001; Morikami et al., 2005). In addition, a sugar responsive cis element that participates in sugar signalling by binding to a WRKY transcription factor in barley (Sun et al., 2003) was also identified in the PvNAS2 promoter. In our continuing work, functional analysis of the PvNAS2 promoter was initiated in order to delineate precisely the cis-acting elements that are responsible for sugar-mediated up-regulation of this gene.
In amide-transporting alfalfa, Shi et al. (1997) showed that the asparagine synthetase gene is expressed in both infected and uninfected cells of the symbiotic zone, with no activity in the outer cortical cells of root nodules. In actinorhizal root nodules of Elaeagnus umbellate, another amide transporter, an asparagine synthetase transcript was detected only in infected cells of the nitrogen-fixing zone (Kim et al., 1999). The present investigation with ureide transporting bean plants, revealed that the expression pattern of PvNAS2 is different from that observed in amide-transporting plants, as the expression was confined only to the outer cortical cells and vascular parenchyma in nodules, but was not observed in the central infected and uninfected cells (Fig. 8B, h–j). The lack of PvNAS2 gene expression in the nitrogen-fixing zone could be due to the reduction in carbon availability because of limiting O2 conditions (Witty et al., 1987).
The present studies with bean demonstrated that metabolizable sugars trigger PvNAS2 induction (Fig. 3C) and concomitantly asparagine production (Fig. 6) in nodules. On the contrary, under such glucose-sufficient conditions ureide content in nodules is reduced (Fig. 6). These results suggest that carbon regulation of asparagine synthetase expression influences the ureide synthesis and transport in bean plants during the symbiotic nitrogen-fixation process. In view of these results, a model is proposed depicting that low O2 levels (resulting because of the nitrogen-fixation activity in nodules; Witty et al., 1987) reduce carbon utilization, leading to the down-regulation of asparagine synthetase expression and the consequent lowering of asparagine biosynthesis (Fig. 10). Under this model, the down-regulation of asparagine synthetase due to low sugar availability creates a favourable situation for the efficient diversion of the amide group of glutamine for purine synthesis through the glutamine-dependent PRPP-amidotransferase, thereby paving a way for ureide production, instead of asparagine synthesis through asparagine synthetase. The absence of asparagine synthetase expression in infected/uninfected cells in the central region of nodules (Fig. 8B, h) probably provides a conducive environment for ureide synthesis in the nitrogen-fixing zone (Smith and Atkins, 2002; Tajima et al., 2004). It is important to mention here that, in bean during nodule maturation, as the nitrogenase activity is induced ureide synthesis increases, becoming the main nitrogenous compound that is transported from nodules to the aerial parts of the plant (Fuentes-Ortiz, 1999).
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The proposed model provides the physiological basis of a mechanism that encourages ureide synthesis in nitrogen-fixing tropical legumes.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Supplementary data can be found at JXB online.
Table S1. PCR primers used for genome walking of the 5' flanking region and amplification of the full length promoter of PvNAS2 gene.
Table S2. Range of 3' UTRs detected in PvNAS2 and its ESTs.
Fig. S1. Nucleotide and deduced amino acid sequences of PvNAS2.
Fig. S2. Comparison of the 3' UTR sequence of PvNAS2 with its ESTs and the orthologue PvAS2.
Fig. S3. Nitrogen-fixing activity of the nodules obtained from the control and sucrose-supplemented Phaseolus vulgaris (cv. Negro Jamapa) plants.
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Acknowledgements |
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We thank Araceli Sánchez for greenhouse assistance, Yadira Gaona and Oswaldo Valdés for technical advice on sugar and RT-PCR analyses, and Dr Fernando Zamudio and Dr Lourival Possani for help in amino acid analysis. This work was supported in part by the funding from Consejo Nacional de Ciencia y Tecnología, México (grant no. G3175-B), and Dirección General del Personal Académico (DGAPA-UNAM; grant no, IN215407).
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Footnotes |
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* These authors contributed equally to this work.
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