Journal of Experimental Botany, Vol. 54, No. 381, pp. 223-237,
January 2, 2003
© 2003 Oxford University Press
Expression of a cyanobacterial sucrose-phosphate synthase from Synechocystis sp. PCC 6803 in transgenic plants
Received 1 July 2002; Accepted 27 August 2002
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia
1 Present address: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Golm, Germany.
2 To whom correspondence should be addressed. Fax: +61 2 6246 5000. E-mail: robert.furbank{at}csiro.au
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Abstract |
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Sucrose-phosphate synthase (SPS) from the cyanobacterium Synechocystis sp. PCC 6803 lacks all of the Ser residues known to be involved in the regulation of higher plant SPS by protein phosphorylation. The Synechocystis SPS is also not allosterically regulated by glucose 6-phosphate or orthophosphate. To investigate the effects of expressing a potentially unregulated SPS in plants, the Synechocystis sps gene was introduced into tobacco, rice and tomato under the control of constitutive promoters. The Synechocystis SPS protein was expressed at a high level in the plants, which should have been sufficient to increase overall SPS activity 2–8-fold in the leaves. However, SPS activities and carbon partitioning in leaves from transgenic and wild-type plants were not significantly different. The maximal light-saturated rates of photosynthesis in leaves from tomato plants expressing the Synechocystis SPS were the same as those from wild-type plants. Tomato plants expressing the maize SPS showed 2–3-fold increases in SPS activity, increased partitioning of photoassimilate to sucrose and up to 58% higher maximal rates of photosynthesis. To investigate the apparent inactivity of the Synechocystis SPS the enzyme was purified from transgenic tobacco and rice plants. Surprisingly, the purified enzyme was found to have full catalytic activity. It is proposed that some other protein in plant cells binds to the Synechocystis SPS resulting in inhibition of the enzyme.
Key words: Carbon partitioning, Lycopersicon esculentum Mill., Oryza sativa L., Nicotiana tabacum L., photosynthesis, rice, sucrose-phosphate synthase, Synechocystis sp. PCC 6803, tobacco, tomato.
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Introduction |
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Sucrose-phosphate synthase (SPS, EC 2.4.1.14) catalyses the penultimate reaction in the pathway of sucrose biosynthesis, in which sucrose-6F-phosphate (Suc6P) is synthesized from UDPglucose (UDPGlc) and fructose 6-phosphate (Fru6P). The Suc6P is then irreversibly hydrolysed by sucrose-phosphatase (SPP, EC 3.1.3.24) to give sucrose (Lunn and ap Rees, 1990a). Studies of SPS-antisense or co-sense suppressed transgenic potato (Solanum tuberosum L.), rice (Oryza sativa L.) and Arabidopsis thaliana (L.) Heynh. plants indicate that SPS makes a major contribution to the control of flux through the pathway of sucrose biosynthesis in both photosynthetic and non-photosynthetic tissues (Geigenberger et al., 1999; Geigenberger and Stitt, 2000; Ono et al., 1999a; Strand et al., 2000).
SPS is a highly regulated enzyme in plants. In addition to allosteric regulation by glucose 6-phosphate (Glc6P) and orthophosphate (Pi), the spinach (Spinacia oleracea L.) leaf SPS is activated in the light and deactivated in the dark by dephosphorylation and rephosphorylation, respectively, of Ser158 (Siegl et al., 1990; Huber and Huber, 1996). In maize (Zea mays L.) leaves, Ser162 has been identified as the phosphorylation site involved in light–dark regulation of the enzyme (Huber and Huber, 1996). There appear to be differences between species in the details of light–dark regulation of SPS by covalent modification (Lunn et al., 1997; Lunn and Furbank, 1999), but equivalent Ser residues are present in almost all known SPS sequences from higher plants (Lunn et al., 1999; Langenkämper et al., 2002). This suggests that there is at least the potential for phosphorylation and regulation of SPS at this site in most plants. There are at least two other known regulatory phosphorylation sites in spinach SPS; Ser424 is involved in osmotic regulation and Ser 229 in binding of 14-3-3 proteins (Toroser and Huber, 1997; Toroser et al., 1998).
Expression of the maize leaf SPS in transgenic tomato (Lycopersicon esculentum Mill.) plants, under the control of the tobacco RbcS promoter, increased SPS activity in the leaves and shifted photoassimilate partitioning away from starch towards sucrose (Worrell et al., 1991). The maize SPS expressed in the transgenic tomato plants was not light–dark regulated, presumably because the tomato SPS-protein kinases were unable to phosphorylate Ser162 in the maize enzyme (Worrell et al., 1991). Some lines of these, and other tomato plants expressing the maize leaf SPS under the control of the cauliflower mosaic virus (CaMV) 35S promoter, showed increased light and/or CO2 saturated rates of photosynthesis, higher relative growth rates and higher fruit yields (Galtier et al., 1993, 1995; Laporte et al., 1997, 2001; Micallef et al., 1995; Murchie et al., 1999; Nguyen-Quoc et al., 1999). Over-expression of SPS in A. thaliana also altered carbon partitioning (Signora et al., 1998) and has been reported to improve photosynthetic performance at low temperature, and to increase freezing tolerance (Strand et al., 2001). Transgenic cotton (Gossypium hirsutum L.) plants with increased SPS activities in fibre cells had improved fibre quality and higher fibre yields than untransformed plants (Haigler et al., 2000), and hybrid poplar (Populus tremula L.xP. tremuloides Michx.) trees with increased SPS activity showed increased photosynthesis and higher growth rates (Mouillon and Hurry, 2001).
Attempts to over-express SPS in other species have been less successful. When the spinach leaf SPS was expressed in tobacco (Nicotiana tabacum L.) plants the enzyme was found to be inactivated by protein phosphorylation (Frommer and Sonnewald, 1995; Toroser et al., 1999), as was the maize SPS expressed in transgenic rice plants (Ono et al., 1999b). Site-directed mutagenesis of the spinach SPS, substituting Ala for Ser158 (S158A), or of the maize SPS (S162A), has been one approach adopted to overcome SPS inactivation by protein phosphorylation (Toroser et al., 1999; Takahashi et al., 2000). However, expression of the maize S162A SPS in transgenic rice did not significantly alter carbon partitioning (Takahashi et al., 2000). Transformation of canola (Brassica napus L.) with the spinach S158A-SPS gene gave some lines with higher SPS activity in the siliques, but not in the leaves (King, 1997). Several of the transgenic canola lines showed co-suppression of the endogenous SPS activity (King, 1997). Similarly, transgenic sugarcane (Saccharum officinarum L.) and rice plants containing the spinach S158A-SPS gene showed no significant increases in leaf SPS activity (CPL Grof, CSIRO Plant Industry, Brisbane, JE Lunn, RT Furbank, unpublished results). The problems of SPS deactivation by protein phosphorylation and co-suppression of the endogenous SPS by introduced plant SPS genes have influenced the search for alternative sources of genes for achieving overexpression of SPS in plants.
The SPS from the cyanobacterium Synechocystis sp. PCC 6803 differs from the plant enzyme in being insensitive to Glc6P and only weakly inhibited by Pi (Lunn et al., 1999). However, an even more interesting feature of the Synechocystis SPS is that it lacks all of the known phosphorylation sites found in SPS from higher plants (Lunn et al., 1999). The Synechocystis sps gene also shows low overall identity with plant SPS gene coding regions; for example, 47% identity with the maize and spinach SPS genes (Lunn et al., 1999).
These observations suggested that the cyanobacterial SPS would not be subject to allosteric regulation or inactivation by protein phosphorylation if it were expressed in transgenic plants. The low nucleotide sequence identity with plant SPS genes also suggested that expression of the Synechocystis sps gene would be less likely to cause co-suppression of the endogenous plant SPS. To investigate the effects of expressing this potentially unregulated SPS in plants, the Synechocystis sps gene has been introduced into tobacco and tomato under the control of the CaMV 35S promoter and into rice under the control of the maize Ubi1 promoter. For each species, several independent lines of plants expressing the cyanobacterial SPS were obtained and the analysis of these plants is presented in this paper.
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Materials
Tomato (Lycopersicon esculentum cv. UC82B) seeds were obtained from Lefroy Valley (Tyabb, Vic., Australia) and rice (Oryza sativa subsp. japonica cv. Taipei 309) seeds were obtained from Dr Narayana Upadhyaya (CSIRO Plant Industry, Canberra). The maize SPS cDNA clone and anti-maize SPS antiserum were obtained from Dr Christine Foyer (IACR-Rothamsted, Harpenden, UK).
Plant growth conditions
Tobacco (Nicotiana tabacum cv. Wisconsin 38) and tomato plants were grown in 25 cm pots of compost containing 5 g of Osmocote slow-release fertilizer per pot. After flowering, tomato plants were also fertilized three times per week with liquid fertilizer (Phostrogen tomato fertilizer). Rice plants were grown in 15 cm plastic pots containing a mixture of soil, perlite, sand, and peat moss (50:25:15:10 by vol) and Osmocote, submerged in water. All plants were grown in a naturally illuminated glasshouse with 28 °C day and 20 °C night temperatures.
Gene construction and plant transformation
Standard cloning procedures were carried out as in Sambrook et al. (1989). For expression of the Synechocystis SPS or maize SPS in tobacco and tomato under the control of the CaMV 35S promoter, the Synechocystis sps (Lunn et al., 1999) or maize SPS (Worrell et al., 1991) coding regions were inserted into the SmaI site of pDH51 (Pietrzak et al., 1986). The resulting 35S-Synsps-tm35S or 35S-ZmSPS-tm35S gene constructs were excised and inserted into the EcoRI site of the binary vector pPLEX502, containing the nptII selectable marker gene under the control of the clover stunt virus Sc1 promoter (Schünmann et al., 2002).
For expression of the Synechocystis SPS in rice under the control of the maize Ubi1 promoter, the sps coding region was inserted between the KpnI and EcoRI sites of pWUbi1.tm1 (Wang et al., 1998). The resulting Ubi1-Synsps-tm1 gene construct was excised and inserted into the HindIII site of the binary vector pWBVec8, containing the hpt selectable marker gene under the control of the CaMV 35S promoter (Wang et al., 1998).
Binary vectors pPLEX502/35S-sps-tm35S, pPLEX502/35S-ZmSPS-tm35S and pWBVec8/Ubi1-sps-tm1 were introduced into Agrobacterium tumefaciens strain AGL1 by triparental mating. The binary vectors were re-isolated from the A. tumefaciens, and the gene coding regions were sequenced to check that no mutations had been introduced during construction of the vectors. Agrobacterium-mediated transformation of tobacco leaf segments was carried out as described by Horsch et al. (1985), of tomato seedling cotyledons as described by Fillatti et al. (1987) and of rice callus as described by Wang et al. (1998). Transformed tobacco and tomato cells were selected on kanamycin-containing media, and transformed rice cells were selected on hygromycin-containing media.
Neomycin-phosphotransferase activity was measured as described in McDonnell et al. (1987). Southern blot analysis was carried out as described in Aoki et al. (2002), using PCR products comprising bases 511 to 1291 of the Synechocystis sps coding region or bases 1500 to 1995 of the maize SPS coding region as probes.
Expression of the Synechocystis SPS in E. coli
The Synechocystis SPS was expressed in E. coli as a fusion protein, linked at the C-terminus by a self-cleaving intein domain to a chitin-binding protein (Lunn, 2002). The fusion protein was purified from E. coli cell extracts by binding to chitin beads, and intein-mediated cleavage of the fusion protein was induced by incubating with 50 mM dithiothreitol (DTT) for 16 h at 4 °C to release the free Synechocystis SPS protein.
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis
Proteins were separated by SDS-PAGE on 7% or 9% (w/v) polyacrylamide gels as described in Laemmli (1970) and either stained with Coomassie Brilliant Blue R-250 or transferred to a nitrocellulose membrane by electroblotting. Membranes were probed with either anti-Synechocystis SPS antibody (1:10 000 dilution in blocking buffer) or anti-maize SPS antiserum (1:500 dilution) as described in Lunn et al. (1999). Anti-phosphoSer, phosphoThr and phosphoTyr antibodies were obtained from Sigma-Aldrich and used at a dilution of 1:500 in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% (v/v) Tween 20, and 0.5% (w/v) bovine serum albumin.
Extraction and assay of SPS
Leaves or leaf discs (0.2–0.5 g) were frozen in liquid N2, and extracted by grinding in a mortar with 1–2 vols of ice-cold extraction buffer (50 mM Tricine-KOH, pH 8.0, 10 mM MgCl2, 1 mM EDTA, 5 mM DTT, 1 mM phenymethylsulphonylfluoride (PMSF), 1 mM benzamide, 1 mM benzamidine, 5 mM 
-aminocaproic acid, 10 µM leupeptin, 10 µM antipain, and 2% (w/v) polyvinylpolypyrrolidone (PVPP)) with about 0.1 g of quartz. The crude extract was centrifuged at 11 600 g for 1 min. A 500 µl aliquot of the supernatant was desalted by passage through a column (bed volume 3 ml) of Sephadex G-25 M (Pharmacia, Uppsala, Sweden), equilibrated with extraction buffer minus PVPP. All procedures were carried out at 4 °C.
SPS was assayed in tobacco and tomato leaf extracts by measuring the UDPGlc, ADPGlc or GDPGlc-dependent synthesis of [14C]Suc6P from [14C]Fru6P as described by Lunn et al. (1997). SPS was assayed in rice leaf extracts by measuring the Fru6P-dependent production of UDP or GDP from UDPGlc or GDPGlc as described by Lunn and Hatch (1997). The assay buffer was 50 mM Tricine-KOH, pH 8.0 and 10 mM MgCl2.
Chlorophyll was determined in methanolic extracts as described by Porra et al. (1989). Protein was determined by the dye-binding assay of Bradford (1976) with bovine 
-globulin as standard.
Photosynthesis and carbon partitioning measurements
Rates of photosynthetic O2 evolution were measured in a leaf disc oxygen electrode (Hansatech Ltd, Kings Lynn, UK), under 5% CO2, as described in Delieu and Walker (1981). Illumination was provided by a 150 W quartz halogen projector lamp attenuated by neutral density filters. The third and fourth fully expanded leaves were chosen for photosynthesis measurements, 2–3 d after flowering had commenced. Two 3.4 cm2 leaf discs were taken from each side of the mid-vein of the two leaves and rates of O2 evolution measured at light intensities of 172 and 2000 µmol quanta m–2 s–1 (providing data in quadruplicate). A single leaf disc was used for the full light response curve of photosynthesis. A representative individual transgenic or untransformed plant, from which sucrose-phosphate synthase activity had been assayed, was chosen for these measurements. Sugars and starch were extracted and assayed as described in Lunn and Hatch (1997).
Immunoaffinity chromatography
Rabbit polyclonal antibodies, raised against the His6-Synechocystis SPS (Lunn et al., 1999), were purified from the antiserum by affinity chromatography on Protein G-Sepharose (Pharmacia) and then coupled to cyanogen bromide-activated Sepharose (Pharmacia) according to the manufacturer’s instructions.
Leaf tissue (2 g) from 35S-Synsps tobacco (1-17) was extracted in 3 ml of ice-cold PBS (10.1 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, and 3 mM KCl, pH 7.4), containing 1 mM EDTA, 1 mM DTT, 2% (w/v) PVPP, and protease inhibitors as described above. All subsequent procedures were carried out at 4 °C. The extract was centrifuged at 35 000 g for 5 min and the supernatant was desalted on a Sephadex G-25M column equilibrated with the PBS extraction buffer minus PVPP. The desalted extract was applied to the antibody-Sepharose column (12 ml) that had been equilibrated with PBS. The column was washed with PBS at a flow rate of 1.4 ml min–1 until the A280 of the effluent returned to the baseline, then washed successively with 30 ml each of 0.1 M glycine (pH 2.5), PBS, 2.5 M KSCN and PBS. Proteins in samples of the original extract (20 µl) and fractions collected from the column (200 µl) were precipitated with acetone (final concentration 80%) and analysed by SDS-PAGE and immunoblotting as described above.
Purification of Synechocystis SPS from transgenic tobacco and rice leaves
Approximately 90 g of deribbed leaf material from 35S-Synsps tobacco or Ubi1-Synsps rice plants were blended for 2 min in a Waring blender in 350 ml of ice-cold extraction buffer (50 mM Hepes-NaOH, pH 7.5, 10 mM MgCl2, 1 mM EDTA, 5 mM DTT, and 2% (w/v) PVPP) containing protease inhibitors as above. After filtering through four layers of cheesecloth the crude extract was centrifuged at 35 000 g for 10 min. The supernatant was decanted and fractionated with polyethylene glycol 8000 (PEG). Protein in the 10–20% PEG fraction was dissolved in 20 ml of buffer A (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 2 mM DTT) containing protease inhibitors. All procedures to this point were carried out at 4 °C and subsequent procedures were carried out at room temperature.
The 10–20% PEG fraction from tobacco was applied to a 5 ml Econo Q anion-exchange column (BioRad, Hercules, CA, USA), equilibrated with buffer A, at a flow rate of 2 ml min–1. Proteins were eluted with a linear salt gradient (0–1 M NaCl over 50 ml), collecting 2 ml fractions. The bulk of the Synechocystis SPS protein eluted between 280–340 mM NaCl. The corresponding fractions were pooled and desalted by passage through a 20 ml column of Sephadex G25M equilibrated with buffer A.
The desalted EconoQ fractions from tobacco or the 10–20% PEG fraction from rice were applied to a 1 ml MonoQ anion-exchange column (Pharmacia), equilibrated with buffer A, at a flow rate of 1 ml min–1. Proteins were eluted with a linear salt gradient (0–1 M NaCl over 30 ml), collecting 0.5 ml fractions after 6 min. The Synechocystis SPS protein eluted between 350–380 mM NaCl. The two peak fractions (1 ml) were combined and concentrated to a volume of about 200 µl using a Millipore Ultrafree-MC centrifugal concentrator (10 000 nominal MW cutoff; Millipore, Bedford, MA, USA). The concentrated protein was applied to a 24 ml Superdex 200 (Pharmacia) gel filtration column equilibrated with buffer A containing 150 mM NaCl, at a flow rate of 0.5 ml min–1. Fractions of 0.2 ml were collected after 20 min. The Synechocystis SPS peak eluted at 27.6 min (14.3 ml).
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Expression of the Synechocystis SPS in tobacco
The Synechocystis sps gene was introduced into tobacco under the control of the constitutive CaMV 35S promoter via Agrobacterium-mediated transformation. Twenty plants, regenerated on kanamycin-containing media, showed neomycin phosphotransferase activity indicating that they contained the nptII selectable marker gene. The plants were screened for expression of the Synechocystis SPS with an antibody raised against the purified His6-SynSPS (Lunn et al., 1999). Ten of the transgenic plants contained an immunoreactive 82 kDa protein, whereas untransformed tobacco plants did not. Figure 1A shows representative results from one transgenic tobacco plant expressing the Synechocystis SPS (lane 2) and one untransformed tobacco plant (lane 1). Southern blot hybridization confirmed that these ten plants contained the 35S-Synsps gene construct, and originated from independent transformation events (data not shown). Of these ten T0 plants, individuals 1-18 and 2-22 were estimated to contain three and at least six copies of the transgene, respectively, but all the other plants contained single transgene loci (data not shown).
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SPS activity was measured in leaf extracts from these ten plants with UDPGlc or GDPGlc as the substrate. Both the endogenous tobacco SPS and the Synechocystis SPS would contribute to the UDPGlc-dependent SPS activity, whereas only the Synechocystis SPS would show GDPGlc-dependent activity (Curatti et al., 1998; Lunn et al., 1999). In this initial screening the UDPGlc-dependent SPS activity in the transgenic tobacco plants ranged from 102–151% of that in wild-type plants (Fig. 2). Low GDPGlc-dependent SPS activity was detected in some of the transgenic plants, but even in plant 1-17 where this was highest, 0.089 µmol min–1 mg–1 Chl, it was less than 12% of the UDPGlc-dependent activity. There was no correlation between GDPGlc-dependent activity and the apparent increase in UDPGlc-dependent activity over wild-type plants. For example, plant 2-5 consistently showed higher UDPGlc-dependent SPS activity than wild-type plants on a chlorophyll basis, but no detectable GDPGlc-dependent activity. These results indicated that, even in the highest Synechocystis SPS-expressing plants, very little of the total SPS activity was attributable to the Synechocystis SPS.
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The immunoblot signal from 20 µg of leaf protein from tobacco plant 1-17 was comparable to that from about 50 ng of the purified His6-SynSPS. From the specific activities of the His6-SynSPS (Table 1), it was estimated that the Synechocystis SPS should contribute about 43 nmol min–1 mg–1 protein UDPGlc-dependent SPS activity and 27 nmol min–1 mg–1 protein GDPGlc-dependent SPS activity to the total leaf activity in this plant. This translates to a total expected SPS activity of about 1.7 µmol min–1 mg–1 Chl with UDPGlc as substrate and 0.7 µmol min–1 mg–1 Chl with GDPGlc as substrate. Clearly, there is a large discrepancy between the SPS activities detected in plant 1-17 and those predicted from these estimates.
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A recovery experiment was carried out in which purified His6-SynSPS was added to a tobacco leaf sample before extraction. SPS activity was assayed in both the supplemented extract and in a control leaf extract without added enzyme. The UDPGlc-dependent SPS activity in the His6-SynSPS-supplemented extract was much higher than in the control extract (Table 2), with the difference in activity corresponding to 109% of that expected from the amount of enzyme added. There was no detectable ADPGlc- or GDPGlc-dependent SPS activity in the control extract, whereas the supplemented extract showed high activity with both substrates, corresponding to 98% and 138% of the expected activity of the added His6-SynSPS (Table 1). In this experiment a leaf extract from 35S-Synsps tobacco line 1-17 showed no detectable ADPGlc- or GDPGlc-dependent SPS activity.
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A homozygous line was established from the primary transformant 35S-Synsps 1-17, for further analysis. T1 progeny from 35S-Synsps 1-17 were screened for expression of the Synechocystis SPS by immunoblotting. Five out of 13 T1 plants showed high level expression of the Synechocystis SPS protein, comparable to the parent plant (data not shown). T2 plants were grown from seed of these five T1 plants and screened by immunoblotting. Two lines showed segregation of Synechocystis SPS-expressing and non-expressing progeny, indicating that their T1 parents were hemizygous. For each of the other three lines, 16 out of 16 plants tested all showed expression of the Synechocystis SPS, indicating that the parental T1 plants were homozygous for the 35S-Synsps gene. One of these lines (35S-Synsps 1-17-2) was selected at random for further analysis. This tobacco line contains a single transgenic locus and expresses the Synechocystis SPS protein at a high level, comparable to the parental plant.
The UDPGlc-dependent SPS activity in leaves of these plants was 0.71±0.16 µmol min–1 mg–1 Chl compared to 0.57±0.13 µmol min–1 mg–1 Chl in leaves from wild-type plants (means ±SD, n=3). Activity in the roots was 11.8±5.5 nmol min–1 mg–1 protein and 6.5±2.1 nmol min–1 mg–1 protein, respectively. Carbon partitioning in the leaves of these plants was determined by measuring the soluble sugar and starch content of leaf samples harvested before sunrise and in the late afternoon. No significant differences were observed in the soluble sugar and starch contents of wild-type and Synechocystis SPS-expressing plants (data not shown). The total amounts of carbohydrate accumulated in the leaves of the wild type and transgenic plants during the day were 41.3 and 42.9 µmol (hexose) mg–1 Chl, and the ratios of starch to sucrose accumulated were 5.5 and 6.2, respectively (n=6).
Expression of the Synechocystis SPS in rice
The Synechocystis sps gene was introduced into rice under the control of the constitutive maize Ubi1 promoter via Agrobacterium-mediated transformation. Fifty-four rice plants were regenerated on hygromycin-containing media from 31 separate calli. Of these, 28 plants showed expression of the Synechocystis SPS protein by immunoblotting with the anti-Synechocystis SPS antibody. Figure 1A shows representative results from one transgenic rice plant expressing the Synechocystis SPS (lane 4) and one untransformed rice plant (lane 3). Southern blot hybridization with probes for the hpt selectable marker gene and the Ubi1-Synsps gene confirmed that all of the plants were transformed. Of the 19 pairs of plants derived from single calli, 17 pairs were judged to be sibling clones from the Southern blot hybridization. Seven of the lines were estimated to contain between 8 and 12 copies of the Ubi1-Synsps gene, and none of these lines showed detectable expression of the Synechocystis SPS protein. Those lines that did express the Synechocystis SPS protein contained between one and four copies of the transgene. Overall, there were 16 independent lines expressing detectable Synechocystis SPS protein. None of these showed significantly different UDPGlc-dependent SPS activity in the leaves compared to untransformed plants (Fig. 3). No ADPGlc- or GDPGlc-dependent SPS activity was detected in any of the plants.
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Expression of the Synechocystis SPS in tomato
These results from tobacco and rice, and the previously reported difficulties in over-expressing SPS in these two species (Frommer and Sonnewald, 1995; Ono et al., 1999b) are in marked contrast with the successful over-expression of SPS in transgenic tomato plants (Worrell et al., 1991; Galtier et al., 1995). This led to the suggestion that, for some reason, tomato is more amenable for achieving over-expression of SPS than some other species. Therefore, it was decided to investigate whether the Synechocystis SPS could be expressed and show activity in tomato. As a positive control, the maize SPS was also expressed in tomato.
The 35S-Synsps and 35S-ZmSPS gene constructs were introduced into tomato (cv. UC82B) via Agrobacterium-mediated transformation. Twenty-eight plants were regenerated on kanamycin-containing medium from transformation with the 35S-Synsps gene construct, of which six showed expression of the Synechocystis SPS protein by immunoblotting. Figure 1A shows representative results from one transgenic tomato plant expressing the Synechocystis SPS (lane 6) and one untransformed tomato plant (lane 5). The strength of the immunoblot signal was comparable to that in 35S-Synsps tobacco line 1-17. Similarly, 27 plants were recovered from transformation with the 35S-ZmSPS gene construct, of which six showed expression of the maize SPS protein by immunoblotting. Figure 1B shows representative results from one transgenic tomato plant expressing the maize SPS (lane 8) and one untransformed tomato plant (lane 7).
SPS assays on leaves of the primary transformants showed that two of the plants expressing the maize SPS protein, numbers 50 and 71, had activities of 1.9 and 1.7 µmol min–1 m–1g Chl, respectively. These activities were about 4-fold higher than that in leaves of wild-type plants, 0.4 µmol min–1 mg–1 Chl. On a protein basis, the activities in plants 50 and 71 were 40 nmol min–1 mg–1 protein, which is about 2-fold higher than in the wild-type plants (22 nmol min–1 mg–1 protein). One of the transgenic plants expressing the Synechocystis SPS protein at a high level, plant 5, showed higher SPS activity on a Chl basis, (1.0 µmol min–1 mg–1 Chl), than the wild-type plant, but slightly lower activity than wild type on a protein basis (17 nmol min–1 mg–1 protein). The primary transformants were recovered from tissue culture over several months, and it was difficult to obtain good replicate samples from plants at different developmental stages for comparison of SPS activities. Therefore, further analysis was carried out on T1 progeny from plants 5, 50 and 71, all grown under the same conditions. Plant 35S-Synsps 5 had a single transgene locus, judged by Southern blot hybridization, and the 35S-ZmSPS 50 and 71 plants had five and three transgene loci, respectively.
Twelve T1 progeny from each of these three parents were screened for expression of the Synechocystis or maize SPS proteins by immunoblotting. SPS activities were measured in all of the immuno-positive plants and several of the immuno-negative plants. None of the T1 progeny from 35S-Synsps plant 5 (5-1 to 5-11) showed higher leaf SPS activity than the wild-type plants (Fig. 4). Also, there were no significant differences in SPS activity between the T1 plants that were expressing the Synechocystis SPS protein (marked by asterisks) and those that were not (Fig. 4). By contrast, six of the T1 plants that expressed the maize SPS protein showed 2–3-fold higher SPS activities than wild-type plants (Fig. 4). SPS activities in three of the 35S-ZmSPS T1 plants that did not express detectable amounts of the maize SPS protein were similar to or lower than those in wild-type plants. One plant, 71-1, that was immuno-positive for the maize SPS showed slightly lower SPS activity than the wild type. However, the activities measured in the three samples from this plant varied considerably (1.35, 0.28 and 0.22 µmol min–1 mg–1 Chl). The fact that two of these samples had only about 25% of the activity in samples from wild-type plants might indicate that there was suppression of the endogenous tomato SPS in some sectors of the leaves of this plant.
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Screening of T2 progeny from the 35S-Synsps 5-8 T1 plant, as described above, indicated that this plant was homozygous for the single transgene locus. However, the T2 progeny from 35S-ZmSPS T1 plants 50-2, 50-8, 50-9, 71-3, 71-5, and 71-6 all showed segregation of maize SPS expression. Photosynthetic activity and carbon partitioning were measured in wild type, 35S-Synsps 5-8 (T2), and 35S-ZmSPS 50-9 (T2) and 71-6 (T2) plants, which showed UDPGlc-dependent SPS activities of 0.68, 0.60, 2.02, and 2.48 µmol min–1 mg–1 Chl, respectively. The rates of photosynthetic O2 evolution at a saturating concentration of CO2 (5% v/v) were the same in wild-type tomato plants and 35S-Synsps plants expressing the Synechocystis SPS protein (Fig. 5). However, 35S-ZmSPS plants expressing the maize SPS showed higher rates of photosynthesis, particularly at saturating irradiance (Fig. 5). For example, the 35S-ZmSPS line 50-9 had 9% and 58% higher rates of photosynthesis than the wild-type plant at irradiances of 172 µE m–2 s–1 and 2000 µE m–2 s–1, respectively (Fig. 5). To investigate photoassimilate partitioning, leaves were sampled from the plants before sunrise and in the late afternoon to measure their soluble sugar and starch contents. The starch contents of the tomato leaves varied enormously, reflected in the large error bars (Fig. 6). Similarly high variability had been found in the leaf starch content of the T1 tomato plants. However, it was clear that leaves from the two independent lines expressing the maize SPS (50-9 and 71-6) had much less starch than either the wild-type plants or the transgenic plants expressing the Synechocystis SPS (5-8) (Fig. 6). The differences between plants in the absolute amounts of sucrose accumulated in the leaves were relatively small.
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Expression of the Synechocystis SPS in E. coli
Even though the Synechocystis SPS protein was apparently expressed at high levels in some lines of all three species, tobacco, rice and tomato, there was little evidence of its activity either in extracts from the plants or from any changes in carbon partitioning. The estimates of expected SPS activity in the 35S-Synsps tobacco were made on the assumption that the enzyme expressed in the plants would have the same specific activity as the His6-SynSPS enzyme expressed in E. coli (Lunn et al., 1999). If the presence of the N-terminal His6-tag were to increase the specific activity of the enzyme, this would lead to overestimation of the expected SPS activity in the transgenic tobacco plants. To test this possibility, the kinetic properties of the Synechocystis SPS without a His6-tag were determined.
The Synechocystis SPS was expressed in E. coli and purified as described in the Materials and methods. The resulting 82 kDa protein differs from the native Synechocystis SPS, and that expressed in the transgenic tobacco plants, in having a single, extra glycine residue at the C-terminus arising from the cloning strategy used to make the gene expression construct.
The Synechocystis SPS expressed in E. coli has a broad pH optimum centred around 8.5, the same as that of the His6-SynSPS, and is not activated by Glc6P. It shows activity with UDPGlc, ADPGlc and GDPGlc, with similar Km values to the His6-tagged enzyme (Table 1). The specific activity with UDPGlc is also the same as that of the His6-SynSPS, although the specific activities with ADPGlc and GDPGlc as substrates are slightly lower (Table 1). From immunoblots, comparing the amount of Synechocystis SPS protein in plant leaf extracts with known amounts of the enzyme expressed in E. coli, the expected activity of the Synechocystis SPS expressed in 35S-Synsps tobacco line 1-17 was estimated to be about 0.17 µmol min–1 mg–1 protein, equivalent to 4 µmol min–1 mg–1 Chl. Similarly, the expected Synechocystis SPS activity in Ubi1-Synsps rice line 43, which had the strongest immunoblot signal, was estimated to be about 0.06 µmol min–1 mg–1 protein, or 2 µmol min–1 mg–1 Chl. These estimates indicated that leaf SPS activity should have been about 8-fold higher than wild type in the highest expressing tobacco lines and over 2-fold higher in the transgenic rice, if the Synechocystis SPS enzyme expressed in the plants were fully active.
Analysis of the Synechocystis SPS expressed in plants
The failure to detect any enzyme activity reproducibly in vitro from the Synechocystis SPS expressed in plants, and the lack of evidence that the enzyme was active in vivo, led to the investigation of the enzyme’s apparent inactivity. Although the Synechocystis SPS does not have any of the known phosphorylation sites of the higher plant enzyme, the possibility that it does have cryptic phosphorylation sites cannot be excluded. Antibodies that recognize phosphorylated-Ser, Thr or Tyr residues cross-reacted with several proteins in leaf extracts from wild-type tobacco and rice plants. However, they did not specifically recognize any 82 kDa protein from the 35S-Synsps tobacco or Ubi1-Synsps rice plants (data not shown).
Although no evidence for phosphorylation of the Synechocystis SPS was found, it was decided to purify the protein from transgenic tobacco plants in order to investigate whether there was some other post-translational modification to the protein that could explain its apparent inactivity. An anti-Synechocystis SPS antibody-Sepharose column was prepared for immunoaffinity purification of the Synechocystis SPS. A desalted leaf extract from 35S-Synsps tobacco (1-17) was applied to the column, which was then washed successively with PBS, 0.1 M glycine (pH 2.5), PBS, 2.5 M KSCN, and PBS. Samples of each fraction were analysed by SDS-PAGE and immunoblotting with the anti-Synechocystis SPS antibody. The Synechocystis SPS was clearly detectable in the crude extract applied to the column, but not in the flow-through fraction (Fig. 7B), indicating that the enzyme had bound to the column. The 0.1 M glycine and subsequent PBS wash fractions did not contain any detectable protein, but the 2.5 M KSCN and final PBS wash fractions did show a single protein band by SDS-PAGE, with a molecular mass of 73 kDa (Fig. 7A). Evidently, this protein was specifically and tightly bound to the anti-Synechocystis SPS antibody column, but it was smaller than the Synechocystis SPS (82 kDa) and was not recognized by the anti-Synechocystis SPS antibody in immunoblots (Fig. 8B). The antibody did not show a reaction with any 73 kDa protein in crude tobacco leaf extracts either (Fig. 1). The 73 kDa protein was unlikely to be derived from antibodies leached from the column as it differed in size from a typical IgG antibody (150–160 kDa) or its component light (24 kDa) and heavy (49 kDa) chains, and it was not recognized by the alkaline-phosphatase conjugated, goat anti-rabbit-IgG secondary antibody used for detection on the immunoblot.
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As immunoaffinity chromatography had failed to purify the Synechocystis SPS, an alternative procedure was developed using PEG precipitation and anion-exchange chromatography on an EconoQ and then a MonoQ column. Initially, fractions containing the Synechocystis SPS protein were identified by immunoblotting. The crude extract and PEG fractions showed UDPGlc-dependent SPS activity, attributable to the endogenous tobacco SPS, and in immunoblots contained a single, 82 kDa protein band that was recognized by the anti-Synechocystis SPS antibody (Fig. 8A). Fractions from the anion-exchange columns that contained the Synechocystis SPS protein showed ADPGlc and GDPGlc-dependent SPS activities, as well as UDPGlc-dependent activity. The EconoQ fractions contained a 54 kDa immunoreactive protein in addition to the 82 kDa protein. In several experiments the 54 kDa protein was the only immunoreactive protein detected in MonoQ fractions with SPS activity, and these did not contain any of the full length, 82 kDa Synechocystis SPS (Fig. 8A). This showed that the 54 kDa truncated form of the Synechocystis SPS must be catalytically active.
The truncated enzyme was purified further by gel filtration on a Superdex 200 column. In this experiment, the fraction from the MonoQ column that was applied to the Superdex 200 column contained only two immunoreactive proteins, the 54 kDa protein and a minor 30 kDa protein (Fig. 8B). Activity eluted from the gel filtration column in a single peak with a retention time corresponding to a molecular mass of 105 kDa. The peak fractions contained two major protein bands by SDS-PAGE, with molecular masses of 54 and 41 kDa (Fig. 8C). The kinetic properties of this highly purified Synechocystis SPS (Superdex 200 fractions 17-19) were investigated. The enzyme showed a broad pH optimum around 8.0 to 8.5, was active in the presence of 10 mM EDTA and in the absence of added Mg2+, and was not activated by 17.5 mM Glc6P. It showed activity with UDPGlc, ADPGlc and GDPGlc as substrates, with similar Km values to the enzyme expressed in E. coli (Table 1). The Vmax activities with all three substrates were higher than for the enzyme expressed in E. coli, but the relative activities with each substrate were comparable, with UDPGlc>ADPGlc> GDPGlc (Table 1). It should be noted that the 54 kDa form of the Synechocystis SPS enzyme probably represented only about half of the protein in these preparations (Fig. 8C), therefore the Vmax values of the pure truncated enzyme would be about 2-fold higher than the values shown in Table 1.
The 54 and 30 kDa immunoreactive proteins (Fig. 8A, B) were presumably cleavage products arising from proteolysis of the 82 kDa Synechocystis SPS during the purification procedure. To investigate this process further, two leaf extracts were prepared from 35S-Synsps 1-17 tobacco, one in the usual way including serine and cysteine-protease inhibitors in the extraction and desalting buffers (see Materials and methods) and the other without protease inhibitors. The two extracts were incubated at 25 °C for 1 h with samples taken at intervals for immunoblotting and SPS assays. In both extracts only an 82 kDa protein was recognized by the anti-Synechocystis SPS antibody, even after incubation at 25 °C for 1 h without protease inhibitors (data not shown). The initial activities in the extracts with and without protease inhibitors were 0.39 and 0.37 µmol min–1 mg–1 Chl, respectively. In the presence of protease inhibitors, 92% of the initial SPS activity remained after 1 h, whereas in the absence of protease inhibitors it had fallen to 49% of the initial activity after 1 h.
The Synechocystis SPS was also partially purified from Ubi1-SynSPS rice plants by PEG fractionation and anion-exchange chromatography on a MonoQ column. Fraction number 14 from the MonoQ column contained the most Synechocystis SPS protein, as judged by immunoblotting (Fig. 9A), and showed SPS activities of 393, 218 and 144 nmol min–1 mg–1 protein with UDPGlc, ADPGlc and GDPGlc as substrates, respectively (Fig. 9B). The peak of UDPGlc-dependent activity was in fraction 13, which showed much lower relative activities with ADPGlc and GDPGlc (Fig. 9C). This activity is largely attributable to the endogenous rice SPS that was not fully resolved from the Synechocystis enzyme. Fractions 13 and 14 showed two minor immunoreactive protein bands of about 60 and 62 kDa in addition to the main 82 kDa protein band (Fig. 9). However, the presence of these bands did not correlate with ADPGlc or GDPGlc-dependent SPS activity, as these activities were higher in fraction 15 than 13, but the former contained only the 82 kDa immunoreactive protein and not the smaller proteins (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The Synechocystis sps gene promised to have several advantages over higher plant SPS genes for achieving over-expression of SPS in transgenic plants, which previous work on tomato had shown could have a beneficial effect on plant productivity. Tobacco and rice were initially chosen for expression of the Synechocystis sps gene as representative plants that accumulate mostly starch (tobacco) or sucrose (rice) in the leaves, and are readily transformable via Agrobacterium. The Synechocystis sps gene was expressed at high enough levels in the plants, judged by immunoblotting, to achieve 2–8-fold increases in SPS activity. Despite the high levels of expression of the Synechocystis SPS, little evidence was found that the enzyme was active. A few of the plants did appear to have higher UDPGlc-dependent activity (Fig. 2), but no ADPGlc or GDPGlc-dependent SPS activity could reproducibly be found in these plants. Such activities would have been definitive evidence that the cyanobacterial enzyme was active, as the endogenous plant SPS is specific for UDPGlc. Recovery experiments with the purified His6-tagged enzyme expressed in E. coli showed that the extraction and assay procedures were suitable for detecting these activities (Table 2). Evidence that the Synechocystis SPS was also inactive in vivo was consistent with the lack of detectable enzyme activity in vitro.
The Synechocystis SPS was also expressed in tomato (Fig. 1), but no reproducible evidence could be found of its activity in tomato either (Fig. 4). Maximal rates of photosynthesis were also unaffected by expression of the Synechocystis SPS (Fig. 5). The leaf starch contents of the tomato plants were extremely variable, which made it difficult to judge whether there was any significant difference in carbon partitioning between wild type and Synechocystis SPS plants (Fig. 6). By contrast, tomato plants expressing the maize SPS had 2–3-fold higher SPS activities, higher rates of photosynthesis and lower leaf starch contents (Figs 4–6), in agreement with previous reports (Worrell et al., 1991; Galtier et al., 1993, 1995; Laporte et al., 1997; Micallef et al., 1995; Murchie et al., 1999; Nguyen-Quoc et al., 1999). None of the plants showed large differences in the amounts of sucrose in their leaves (Fig. 5). Even if more sucrose were synthesized, the high acid invertase activity present in tomato leaves (Gao et al., 1998) would probably limit the amount of sucrose accumulated (Winter and Huber, 2000). However, from the rates of photosynthesis and decreased starch accumulation in the 35S-ZmSPS plants, it could be inferred that a higher proportion of photoassimilate was being exported from the leaves, reflecting greater partitioning into sucrose.
The lack of detectable Synechocystis SPS activity even in tomato, where we and others have been able to increase SPS activity by expressing the maize SPS, suggested that there is a general problem with expression of the cyanobacterial enzyme in plants. No evidence was found that the enzyme was phosphorylated on any Ser, Thr or Tyr residues, which was one possible explanation for the lack of activity in plants. Therefore, it was decided to purify the enzyme from the transgenic tobacco plants for further investigation. The initial attempt at purification by immunoaffinity chromatography yielded a 73 kDa protein, which although evidently tightly bound to the antibody-Sepharose column, was not recognized by the Synechocystis SPS antibody (Fig. 7). After this puzzling result, more conventional procedures were used to purify the enzyme.
Surprisingly, the Synechocystis SPS purified from leaves of transgenic tobacco plants showed full SPS activity with UDPGlc, ADPGlc and GDPGlc as substrates (Table 1) with a broad pH optimum around 8.0–8.5, and was not activated by Glc6P. These distinctive properties are characteristic of the Synechocystis SPS (Lunn et al., 1999). Similar results were also obtained from rice (Fig. 9). These results conclusively showed that the Synechocystis SPS protein expressed in the transgenic plants was inherently active. The lack of activity in crude extracts must, therefore, be due to inhibition of the enzyme by some factor in the plant cells, which almost certainly also inhibited the enzyme in vivo.
No Synechocystis SPS activity was detectable even in desalted extracts, so the inhibitor was unlikely to be a low molecular weight compound. The most likely alternative seemed to be another protein, and the unexpected result from the immunoaffinity chromatography experiment suggested a possible candidate. If the 73 kDa protein purified in this experiment were bound to the Synechocystis SPS, this could explain why it was retained on the column, but not recognized by the anti-Synechocystis SPS antibodies. If plant cells do contain a protein that binds and inhibits the Synechocystis SPS, an inhibition of the enzyme might have been expected in the recovery experiment (Table 2). However, the rate of association between the putative inhibitory protein and the Synechocystis SPS could be too slow to be seen in the time scale of the recovery experiment.
Another interesting observation from the purification of the Synechocystis SPS from tobacco was the appearance of truncated forms of the enzyme. The appearance of the 54 kDa and 30 kDa immunoreactive proteins coincided with the disappearance of the 82 kDa form of the enzyme, suggesting that the Synechocystis SPS had been proteolytically cleaved (Fig. 8A, B). The 54 kDa-protein was the only immunoreactive protein in some fractions, but these contained high SPS activity showing that the truncated enzyme was catalytically active.
The Synechocystis SPS, like the higher plant enzyme, contains two domains; a larger, N-terminal glucosyltransferase domain and a smaller, C-terminal domain that shows similarity to SPP (Lunn et al., 2000). The glucosyltransferase domain of the Synechocystis SPS contains about 464 amino acid residues while the SPP-like domain contains about 256 residues, with predicted molecular masses of 52 and 29 kDa, respectively. These values are very similar to the 54 and 30 kDa proteins derived from the Synechocystis SPS, suggesting that the latter had been cleaved close to the junction between the two domains. This implies that the 54 kDa protein fragment contains the intact glucosyltransferase domain, consistent with it retaining SPS activity.
The truncated form of the enzyme was observed in several purification experiments from tobacco, and seemed to be linked to the appearance of SPS activity. An experiment to test this correlation, by incubating a crude leaf extract at 25 °C without protease inhibitors to favour proteolysis of the Synechocystis SPS, was inconclusive, because the protein was not cleaved. However, when the enzyme was partially purified from rice leaves there was no correlation between the presence of truncated Synechocystis SPS protein and enzyme activity (Fig. 9). This indicates that it was purification of the enzyme not proteolytic cleavage that allowed activity to be revealed, and that the latter was coincidental and not essential for seeing activity.
From these results, it is proposed that the Synechocystis SPS expressed in plants is inherently active, but is inhibited in vivo by binding of an endogenous plant protein, possibly the 73 kDa protein purified on the antibody-Sepharose column. Removal of the putative inhibitor protein during purification of the Synechocystis SPS reveals its activity, and perhaps allows proteolytic cleavage of the enzyme to give the truncated 54 kDa form that retains catalytic activity. This form of the enzyme appears to be dimeric, whereas the intact enzyme is monomeric.
As mentioned in the Introduction, Ser229 in the spinach leaf SPS is the phosphorylation site involved in binding of 14-3-3 proteins (
30 kDa) to the enzyme (Toroser et al., 1998). When A. thaliana suspension cells were starved of sugar, the amount of 14-3-3 protein bound to SPS decreased and the enzyme was proteolytically cleaved, but still retained catalytic activity (Cotelle et al., 2000). The truncated SPS had a molecular mass of about 90–95 kDa, which is very similar to the predicted size of the N-terminal, glucosyltransferase domain of a typical higher plant SPS. This suggests that the enzyme had been cleaved close to the junction between the glucosyltransferase domain and the C-terminal, SPP-like domain (Lunn et al., 2000). There are several reports of preparations of SPS purified from plants containing proteolytic fragments of about 90 and 30 kDa (Lunn and ap Rees, 1990b; Bruneau et al., 1991). The observation that the Synechocystis SPS, like the higher plant enzyme, is also prone to specific cleavage in this junction region suggests that proteolytic processing of SPS to remove the SPP-like domain could be a widespread mechanism for regulating SPS activity. The significance of this for control of sucrose synthesis is unclear and warrants further investigation.
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Acknowledgements |
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We thank Dr Narayana Upadhyaya, Dr Petra Schünmann and Ms Judy Gaudron (CSIRO Plant Industry, Canberra) for their help with the rice transformation, and Dr Christine Foyer (IACR-Rothamsted, Harpenden, UK) for the generous gifts of the maize SPS cDNA clone and anti-maize SPS antiserum .
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