Journal of Experimental Botany, Vol. 54, No. 383, pp. 749-755,
February 1, 2003
© 2003 Oxford University Press
Heterogeneity of sucrose synthase genes in bean (Phaseolus vulgaris L.): evidence for a nodule-enhanced sucrose synthase gene
Received 30 September 2002; Accepted 25 October 2002
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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Sucrose synthase (SS), the key sucrose hydrolytic enzyme (EC 2.4.1.13), plays an important role in N2-fixing nodule metabolism. It has also been proposed that N2 fixation in soybean nodules could be mediated by the potential to metabolize sucrose. The isolation and characterization of a nodule-enhanced SS full-length cDNA clone from the bean Phaseolus vulgaris is reported here. Southern blot analysis indicated that there are at least two SS genes in beans. Using a 3' specific probe from this SS cDNA clone, it was possible to identify a nodule-enhanced SS gene (PvSSn), which is expressed almost exclusively in nodules. A second gene (PvSS), which is expressed in all tissues tested, was detected using a coding region probe. Nodule-enhanced PvSSn transcript levels, but not the enzyme activity or protein amount, is reduced during nodule development. These data indicated that this reduction could be due to a limitation on the carbon availability in the nodule. PvSSn expression is reduced in the asparagine-treated nodules. By contrast, PvSSn transcript levels in nodules increased in the presence of glutamine, allantoin and allopurinol. This result suggests a relationship between ureide transport and SS regulation and could help in understanding why the ureide transport mechanism is activated during nitrogen fixation in bean.
Key words: Bean, nitrogen fixation, nodules, Phaseolus vulgaris, sucrose synthase genes.
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Introduction |
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A major part of the carbon produced during photosynthesis is channelled into the synthesis of sucrose which is central to plant growth and development. Sucrose is hydrolysed by sucrose synthase (SS) (Morell and Copeland, 1985), a homotetrameric enzyme that catalyses the reversible, UDP-dependent cleavage of sucrose into UDP-Glc and fructose (Akazawa and Okamoto, 1980). These resulting hexoses directly or after conversion into other activated sugars are used in diverse pathways that are critical for plant cell function (Delmer and Amor, 1995; Chourey and Nelson, 1976; Ruan et al., 1997).
Although control of SS activity involves transcriptional regulation (Fu and Park, 1995; Fuller and Verma, 1984), evidence has been presented for post-transcriptional regulation involving serine phosphorylation and a Ca2+-dependent protein kinase (Huber et al., 1996; Zhang and Chollet, 1997). In addition, the expression of SS genes has been shown to be cell-specific, developmentally regulated or regulated by tissue carbohydrate status (Koch et al., 1992; Ruan et al., 1997).
In monocotyledonous and dicotyledonous species, SS is encoded by two differentially expressed non-allelic loci sus1 and sus2 (Fu and Park, 1995). In rice plants even a third SS gene has been identified (Huang et al., 1996). SS has been documented and studied in several legume species (Thummler and Verma, 1987; Küster et al., 1993; van Ghelue et al., 1996; Gordon and James, 1997; Fedrova et al., 1999), however, in legumes only a single SS gene had been identified until recently. Barratt et al. (2001) described different isoforms of SS in pea.
In the legume–Rhizobium symbiosis, nodule development and functioning are primarily dependent on the import and metabolism of sucrose. This disaccharide provides the energy and C skeletons for nitrogen-fixation, the assimilation of ammonia and the export of nitrogen-fixation products (Gordon, 1995; Streeter, 1995). Photosynthetic sucrose is exported via the phloem and unloaded in the nodule cortex where it diffuses into the infected cells to be metabolized. The products of sucrose catabolism (mainly malic acid) are then used by the bacteroids to fuel nitrogen fixation (Vance and Heichel, 1991; Gordon, 1995). Ammonia is exported from the bacteroids into the plant cell cytosol where it is assimilated, and directed to the synthesis of amino acids and/or ureides for subsequent export from the nodule. In soybean root nodules a major nodule-enhanced protein (nodulin-100) has been identified as a SS (Thummler and Verma, 1987). The levels of SS protein and RNA were higher in the N2-fixing nodules than in the other tissues of the legume plants. Its enhanced expression during N2 fixation is consistent with its role in nodule function. This is in agreement with the finding that SS expression is down-regulated in response to stress that simultaneously reduces N2 fixation (González et al., 1995; Gordon et al., 1997), suggesting a role for nodule SS activity in supplying carbohydrate for N2 fixation (Gordon et al., 1999). In pea nodules, however, ABA induces a decline in nitrogen fixation, but does not affect sucrose synthase activity, suggesting that N2 fixation is not entirely dependent on C supply solely made available by the activity of nodule SS (González et al., 2001).
In this study evidence is presented for the existence of two SS genes in bean (Phaseolus vulgaris L.). One of these genes is expressed almost exclusively in nodules (PvSSn) and the other is expressed in all the tissues tested (PvSS). These data suggest that the expression of the PvSSn gene is dependent on C availability and mediated by the status of nitrogen metabolism components in bean nodules.
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Materials and methods |
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Plant growth and treatments
Phaseolus vulgaris cv. Negro jamapa seeds (ProNaSe, México DF) were surface-sterilized in 20% sodium hypochlorite solution for 10 min, and germinated on moist sterile filter paper. Three-day-old seedlings were transferred to pots containing vermiculite (5 plants per pot) and then inoculated with Rhizobium etli strain CE3. Plants were grown in a naturally lit greenhouse as reported by Ortega et al. (1992). Nodules were harvested from 11–21 d after inoculation; while other tissues were harvested from 11 and 21-d-old plants. Three-day-old roots were detached from the cotyledon and used to analyse SS expression in response to carbohydrates. All tissues were immediately frozen in liquid nitrogen and stored at –70 °C until use. Excised roots incubated in the absence of sugars for 24 h were used as the control.
Experimental treatments to assess the effects of different N compounds were imposed on 17-d-old plants. Plants were grouped randomly into five treatment sets; control, Asn (10 mM), Gln (10 mM), allantoin (10 mM), and allopurinol (2 mM). Solutions of the nitrogenous compounds were prepared in Hoagland nutrient medium and 200 ml of each solution was applied daily to each pot for 4 d before the tissues were harvested for analysis. Nodules for RNA extraction were frozen in liquid nitrogen and stored at –80 °C. The plants irrigated only with Hoagland solution served as control.
Protein extraction and enzyme assay
Bean tissues were homogenized in a chilled mortar and pestle with extraction buffer (50 mM Hepes, pH 7.8, containing 1 mM EDTA, 20% [v/v] glycerol, 5% [v/v] ethylene glycol, 5 mM dithiothreitol, and 1 mM phenylmethylsulphonyl fluoride). The homogenate was centrifuged for 15 min at 15 500 g at 4 °C to obtain the soluble protein fraction. SS activity was measured spectrophotometrically monitoring the disappearance of NADH at 340 nm as described by Morell and Copeland (1985). Protein was measured by the method of Bradford (1976).
SDS-PAGE and Western blotting
Soluble proteins (20 µg per lane) were separated on 10% SDS polyacrylamide gels and transferred onto nitrocellulose membranes (Hybond-C extra, Amersham). Blots were blocked in 5% (w/v) commercial non-fat dried milk in TBS (10 mM Tris-HCl, pH 8.0, 150 mM NaCl with 0.1% Tween 20). Blots were probed with antibodies specific for SS (kindly provided by AJ Gordon, Institute of Grassland and Environmental Research, UK). SS protein was detected using a secondary antibody conjugated to alkaline phosphatase, according to the manufacturer’s instructions (Boehringer).
Analysis of RNA
For northern analysis, RNA was extracted from 0.2 g of frozen cotyledon, stem, leaf, root or nodule tissue using an RNA extraction kit (BIO-101). The RNA (10 µg) was denatured in 50% formamide, 17% formaldehyde and 10% MOPS buffer (200 mM MOPS [pH 7.0], 50 mM Na-acetate, and 1 mM EDTA) at 65 °C for 5 min. Ten µg total RNA were separated on a 1.2% agarose gel containing 2.2 M formaldehyde in MOPS buffer and transferred to positively-charged nylon membranes (Hybond-N+, Amersham) by downward capillary transfer in 5x SSC+10 mmol l–1 NaOH.
After a 30 min prehybridization (300 mM Na2HPO4 pH 7.2, 7% SDS), the blot was hybridized for 24 h at 65 °C with 32P-labelled SS probe. After stringent washing, radioactive membranes were exposed to X-Ray film (Kodak) overnight at –70 °C.
Isolation of PvSS cDNA
A nodule-specific cDNA library from P. vulgaris root nodules was screened for full-length SS clones by probing with a 500 bp fragment amplified by PCR with a forward primer (5'-GCACAGTGCCTCCTGAAACC) and a reverse primer (5'-CTTGAGATCCACTTGCGAAC) derived from sequence analysis.
The PvSS cDNA inserts from positive clones were subcloned into pBluescript SK(+) vector as EcoRI-XhoI fragments. An isolated clone was sequenced (Medigenomix, Munich, Germany) and shown to contain the entire SS coding region based on sequence analysis using the GCG software package (accession number AF315375).
Plant DNA extraction and Southern blot
Genomic DNA was isolated from leaves of 5-d-old seedlings using a DNA isolation kit (GENTRA, Minnesota, USA). 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 nylon membranes (Hybond-N+, Amersham). Gene-specific DNA fragments from the coding region (HindIII-XhoI, 2154 bp); (5'-probe) and from the 3' region (XhoI/XhoI, 350 bp; 3' UTR-probe) of the PvSS cDNA were labelled with 32P by random priming (multiprime DNA-labelling kit, Amersham). The blots were prehibridized 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 0.1xSSC with 0.1% (w/v) SDS at 65 °C for 30 min each, and exposed to Kodak X Omat films.
Soluble carbohydrate analysis
Glc, Fru, and Suc were determined in enzymic reactions coupled to the production of NADH, as described by Gordon et al. (1999).
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Results |
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Isolation and characterization of a cDNA encoding PvSSn
A full-length cDNA clone encoding PvSSn (named PvSSn) was isolated from a nodule cDNA library. The PvSSn cDNA clone is 2738 bp long containing a 2418 bp coding region starting at 51 bp and the TGA codon is at the position 2469–2471 bp. The clone also contained 269 bp 3' UTR with a polyadenylation signal AATAA at 165 bp upstream of the poly-A tail. This signal is highly homologous to the consensus sequences of the plant polyadenylation signal (AAUAAAA) (Joshi, 1987). The PvSSn open reading frame encoded an 805 amino acids protein with a predicted molecular mass of 92 kD (Fig. 1). Compared to other reported legumes SS proteins, PvSSn is 95, 94, 91, and 90% identical to those of Vigna, Glycine max, Vicia faba, and Medicago sativa, respectively. Compared to maize, PvSSn was less than 75% identical to either SS isoforms SS1 or SS2. The amino acid sequence of PvSSn showed many conserved motifs as in SS genes of different plants. The N-terminus of PvSSn contains a Ser11 phosphorylation site conserved in most of the SS cDNAs from different species (Fig. 1). As has been reported for maize and potato, PvSSn showed two transmembrane domains (residues 269–289 and 675–695). The amino acid sequence of the first domain is identical to the potato hydrophobic domain I (Winter et al., 1997) (Fig. 1). The second domain is identical to the potato domain II in its first 18 residues; however, in PvSSn, the last two amino acids were Cys and Asn instead of Asn and His as in tobacco.
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SS genes in the genome of Phaseolus vulgaris
To assess whether there may be other isoforms of SS in beans, blots of genomic DNA digested with several restriction enzymes were probed with either the 5'-probe, or the 3' UTR-probe of the PvSSn cDNA clone (see above). Blots with the 5'-probe revealed that bean contains at least two genes encoding SS (Fig. 2A). On the other hand, hybridization with the 3' UTR-probe revealed only one of the two SS genes (Fig. 2B). The Southern analysis indicated that the 3'-UTR probe is able to detect a specific SS gene, and that the 5'-probe is a non-specific probe, which is able to detect at least two SS genes in bean.
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Regulation of SS in bean plants
Temporal and spatial patterns of expression of the two PvSS genes were revealed by hybridization of RNA isolated from cotyledon, stem, leaf, root, and nodule tissues of 11 and 21-d-old plants with the 5'-probe and the 3'-UTR probe (Fig. 3). Hybridization with the 5'-probe shows a 2.8 kb transcript, which is expressed in all tissues tested, but predominantly in nodules (Fig. 3). Levels of expression were lower in 11-d-old stems and at very low levels in stem and roots of 21-d-old plants (Fig. 3). The 3'-UTR probe revealed a transcript, which is almost exclusively expressed in nodules. This result suggests that, in bean nodules, there is a nodule-enhanced SS gene. Analysis of SS activity in different bean tissues, revealed that SS activity is in accordance with the amount of SS protein and with the transcript levels detected in the plant organs analysed, except for 21-d-old leaves where SS transcript levels did not correlate with SS activity and amount of protein. The highest SS activity, protein amount and mRNA levels were found in young (11 d) rather than in mature (21 d) plants, supporting a main role of this enzyme in sink rather than in source tissues and in young rather than in old roots and nodules.
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Regulation of SS in bean nodules
Expression of PvSS genes during nodule development was also investigated using both 5' and 3'-UTR probes. As shown in Fig. 4, PvSS transcript levels remained relatively constant when the 5'-probe was used. When the 3'-UTR probe was used, PvSSn transcript levels declined from days 13 to 21 after inoculation. In contrast to the decline in the transcript levels of the PvSSn gene, SS protein and activity increased slightly from day 11 to day 18 after inoculation (Fig. 4). The discrepancy between the reduction in PvSSn transcript levels and the increase in SS protein and activity observed during nodule development, could be explained by the presence of a second SS isoform in the nodule.
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Metabolic regulation of PvSSn gene
It has been reported that the expression of SS in nodules is regulated by the availability of carbon (Koch, 1996). These results show a reduction of sucrose, glucose and fructose concomitant with the reduction in PvSSn transcript levels during nodule development. In order to assess if this reduction in PvSSn transcript levels was due to a carbon limitation, detached roots from cotyledons were grown for 24 h in darkness in the presence of different sugar concentrations. As shown in Fig. 5A, both glucose and sucrose induced the expression of the PvSSn gene. However, lower concentrations of sucrose than glucose induced maximal transcription of this PvSSn, indicating that this sugar could be a main regulator of SS expression in bean plants.
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SS activity has been reported to be closely reIated to N2 fixation (Gordon et al., 1997). In order to assess if the bean SS expression is under the regulation of a nitrogen compound, nodulated plants were grown for 4 d in the presence of different nitrogenous compounds and analysed for SS gene expression (Fig. 5). The results revealed that asparagine inhibits the expression of the PvSSn gene (Fig. 5B), while glutamine and allantoin marginally induce the expression of this gene. Surprisingly, PvSSn transcript levels increased when plants were treated with allopurinol, which is an inhibitor of the synthesis of ureides. This result suggests that a metabolite in the purine and/or a component from the initial steps of the ureide biosynthetic pathway could play a positive role in the expression of PvSSn gene.
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Discussion |
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The results demostrated that SS in bean is encoded by at least two genes. Using a 3'-UTR probe, it was possible to define one of the two SS genes as nodule-enhanced (PvSSn). It was not possible to obtain a specific probe to study the expression pattern and regulatory characteristics of the second PvSS gene, which is expressed in various plant organs including nodules. A comparison of the nucleotide sequences of both bean SS genes showed a very high similarity in their coding regions. This may explain why, for most of the legumes studied, only one SS has been described (Heim et al., 1993). Nevertheless, the recent findings describing distinct isoforms of SS in Pisum sativum (Barratt et al., 2001) as well as this study’s results suggest the existence of a SS gene family in legumes.
The presence of two hydrophobic domains and of a phosphorylation site at residue Ser11 suggest that the phosphorylation state of the PvSSn protein could be part of the membrane associated process, as has been proposed for different plant SS (Amor et al., 1995; Carlson and Chourey, 1996; Winter et al., 1997). However, Komina et al. (2002) suggested that the phosphorylation state of SS protein in soybean nodules is not clearly related to the intracellular partitioning between the cytoplasm and the plasma membrane. A membrane-associated form of SS has been postulated to play a role in the synthesis of cellulose and callose (Amor et al., 1995). Recent studies in pea embryos, however, showed that Sus1 provides carbon from sucrose preferentially to starch synthesis rather than for cellulose production (Barratt et al., 2001). In bean, studies are needed to clarify the function of the two different isoforms of SS in plant metabolism.
Northern analysis indicated that PvSSn is expressed as a nodule-enhanced gene (Fig. 3). The pattern of expression of SS isoforms in P. sativum revealed that Sus1 expression is high in nodules as well as in roots and embryos. Nevertheless, similar to the maize sus1 gene (Koch et al., 1992) and the potato sus4 gene (Fu and Park, 1995), the expression of PvSSn could be induced in bean roots after supplementation with sugars (Fig. 5). In addition, these results also showed the reduction in PvSSn transcript levels concomitant with a reduction of sugar (sucrose, glucose and fructose) contents during nodule development (Fig. 4). These results are in agreement with those reported by Koch (1996) indicating that SS transcript levels are modulated by carbon availability.
As bean is an ureide transporting plant when nodulated, the inhibitory effect of asparagine on the expression of PvSSn, could suggest that the reduction of the export of amides (particularly asparagines) allows a higher expression of PvSSn, resulting in a better carbon supply for maintaining nitrogen fixation. Nevertheless, further studies on this aspect could help in understanding why bean and other legumes switch from amide to ureide transport during symbiosis.
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Acknowledgements |
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We are grateful to Dr Pallavolu M Reddy for critically reviewing the manuscript and to MC Lourdes Blanco for providing the
gt-22a library. We thank Araceli Sánchez for greenhouse assistance. This project was partially supported by the financial assistance from the Consejo Nacional de Ciencia y Tecnología (CONACyT) grant G31751-B.
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