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Characterization of native and yeast-expressed tomato fruit fructokinase enzymes
Phytochemistry 58 (2001) 841–847 www.elsevier.com/locate/phytochem
Characterization of native and yeast-expressed tomato fruit fructokinase enzymes Marina Petreikov, Nir Dai, David Granot, Arthur A. Schaffer* Department of Vegetable Crops, Agricultural Research Organization, Volcani Center, Bet Dagan, 50250, Israel Received 8 April 2001; received in revised form 16 July 2001
Abstract Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Lycopersicon esculentum Mill.; Hexokinase; Substrate inhibition; Gene product
1. Introduction The developing tomato fruit sink metabolizes translocated suc via either invertase or suc synthase. The path of sucrose catabolism is developmentally regulated: the sucrose synthase pathway is associated with the young fruit which transiently accumulates starch, while the invertase pathway is associated with the later stages of fruit development (Ruan and Patrick, 1995; Ho, 1996; Schaffer and Petreikov, 1997a). In addition to changes in the biochemical pathway, the transition between the two developmental stages is accompanied by changes in the translocation pathway, from a symplastic to an apoplastic one, as well (Ruan and Patrick, 1995; Ho, 1996). Irrespective of the initial enzyme in the pathway, free fru accounts for one half of the imported suc and its phosphorylation is requisite for its further metabolism. Fru phosphorylation may be carried out either by * Corresponding author. Tel.: +972-3-9683646; fax: +972-39669642. E-mail address: [email protected] (A.A. Schaffer).
hexokinases (EC 2.7.1.1), which can phosphorylate both glucose and fructose, or by specific fructokinases (EC 2.7.1.4). The irreversible fructokinase reaction is likely to play an important role in determining carbon flux in sink tissue (Viola, 1996). The activity pattern of fructokinase in developing tomato fruit, but not hexokinase, parallels the transient starch content in the fruit (Schaffer and Petreikov, 1997a). Fructokinases have been characterized from a number of plant tissues (see recent review by Pego and Smeekens, 2000). It is not uncommon to find two or three isoforms upon separation by ion-exchange chromatography (i.e. potato tuber, Gardner et al., 1992; Renz and Stitt 1993) which may also show tissue and developmental specifity (Renz et al., 1993). Some fructokinases show substrate inhibition by fructose (i.e. tomato, Schaffer and Petreikov, 1997b; potato, Gardner et al. 1992; Renz and Stitt, 1993) which may be physiologically significant in the case of tomato fruit which contains high fructose levels (Schaffer and Petreikov, 1997b). Fructokinases can also be characterized by their nucleotide specificity; Huber and Akazawa (1985) reported a fructokinase utilizing UTP while most other
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00331-4
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reported fructokinases show relative specificity to ATP. FKs can also be distinguished by the characteristic of inhibition by excess Mg ions. The fructokinase from pollen grains (Nakamura et al., 1991) and from soybean nodules (Copeland and Morell, 1985) was inhibited by Mg concentrations above 1–2 mM while that of pea seeds was not (Turner et al., 1977). A fructokinase from tomato fruit similarly showed the phenomenon of Mg inhibition (Schaffer and Petreikov, 1997b). Tomato fruit fructokinases have recently attracted increasing attention due to their possible role in transient starch accumulation (Schaffer and Petreikov, 1997a; Kanayama et al., 1997, 1998), in addition to possible physiological roles in hexose accumulation and sugar sensing (Pego and Smeekens, 2000). MartinezBarajas and Randall (1996) separated two isoforms of fructokinase from tomato fruit, which showed similar sensitivity to inhibition by fructose and similar nucleotide specificity. However, Kanayama et al. (1997) cloned two fructokinase genes from tomato with different expression patterns, as well as different kinetic characteristics of the expressed protein. One of the genes (LeFRK2) which is expressed primarily in the early stages of fruit development yields a gene product which, when expressed in yeast shows inhibition by fructose (Kanayama et al., 1998). The other gene (LeFRK1) is expressed throughout fruit development (Kanayama et al 1997) and its yeast-expressed gene product is not inhibited by fructose (Kanayama et al., 1998). The LeFRK2 gene was hypothesized to be responsible for the fructokinase activity associated with transient starch accumulation (Kanayama et al., 1998) while the LeFRK1 gene was suggested to play a housekeeping role in carbohydrate metabolism throughout the plant, supplying fru6P for glycolysis. In light of these recent data we reinvestigated fructokinase activity in developing tomato fruit. We report here three FK isoforms, which can be distinguished one from the other on the basis of fru and Mg inhibition and nucleotide specificity. Furthermore, we attempt to relate the two known LeFRK gene products to the FK isoforms present in the tomato fruit.
observed using two assay methods. One assay contained low fru (1.0 mM) and low Mg (0.5 mM), with which the FKI enzyme showed maximal activity. The second assay contained high fru (10 mM) and high Mg (3 mM), which gave maximal activities for FKII. The FKIII enzyme, which eluted only under high ionic concentration, showed approximately equal activities with both assays. 2.1.2. Activity during development The total FK activity in crude extracts of immature (15 DAA) and ripe fruit pericarp is presented in Table 1. Recovery experiments indicated that differences in extractable activity between the different stage fruit were not artifactually due to inhibitory substances in the mature fruit. Similar recovery of a known amount of purified FKI (from the MonoQ fractions, as described further on) was obtained from both fruit developmental stages (not shown). Activity with both assay systems is higher in the young fruit and sharply declines in the more mature fruit. At both stages of fruit development activity is higher with the low fru/low Mg assay system, indicating that even in the mature fruit the major activity is inhibited by excess fructose and Mg. A comparison of the chromatograms of the pericarp fructokinase enzymes from immature (15 DAA) and ripe fruit stages (Fig. 2) indicates that the three peaks of fructose phosphorylation activity were observed in both stages. The enzyme first eluted (FKI), assayed under low substrate conditions, remained the dominant peak of activity even in the ripe fruit. The FKII enzyme was present in the ripe fruit but generally was the least active of the three FKs. 2.2. Characterization of the three tomato fruit FK enzymes and comparison with the two yeast-expressed LeFRK (LeFRK-Y) gene products In order to determine the relationships between the native FK enzymes and the products of the two LeFRK genes, enzyme extracts from mutant yeast lacking endogenous hexose kinase activity and expressing each of the two described LeFRK genes were separated on
2. Results 2.1. Fructokinase isoenzymes during fruit development
Table 1 Fructokinase activity in immature and mature tomato fruit pericarp Maturity stage
2.1.1. Separation on HPLC-ion exchange Hexose phosphorylation activity from young tomato fruit were separated by HPLC-ion exchange chromatography (Fig. 1). We observed three peaks of activity with fructose as substrate, as well as two additional peaks of activity with glucose (termed HKI and HKII). The three fructose phosphorylating peaks (termed FKI, FKII and FKIII, according to the order of elution) were
Fructokinase activity (nmol g
1
fw min 1)
Assay 1a
Assay 2b
Immature
184+25
85+8
Mature
63+8
27+1
a
Assay 1 contained low fru (1.0 mM) and low Mg (0.5 mM). Assay 2 contained high fru (10 mM) and high Mg (3 mM). Data are averages+S.E. of 4 (immature) and 7 (mature) separate extractions from individual fruit. b
M. Petreikov et al. / Phytochemistry 58 (2001) 841–847
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Fig. 1. HPLC-ion exchange (MonoP) separation of hexose kinase activity in immature tomato fruit pericarp. Assay 1 contained low fru (1.0 mM) and low Mg (0.5 mM). Assay 2 contained high fru or glu (10 mM) and high Mg (3 mM).
Fig. 2. HPLC-ion exchange (MonoQ) separation of fructokinase activity in immature (A) and mature (B) tomato fruit pericarp. Assays are as in Fig. 1. Note the different scales in the two chromatograms.
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MonoQ ion chromatography under the same conditions as the tomato extract (not shown). The partially purified native FK enzymes and the yeast-expressed enzymes were partially characterized and compared as described below. 2.2.1. Hexose specificity and inhibition by fru All three native fruit enzymes were specific for fru, with no discernible activity with glu or mannose (not shown). Of the three enzymes FKII had the lowest affinity for the substrate (Fig. 3a). Both yeast-expressed enzymes were also specific for fru, with no discernible activity with glucose or mannose. The LeFRK1-Y enzyme has an affinity pattern for fru analogous to FKII (Fig. 3b). Both FKI and FKIII from tomato fruit show the characteristic of substrate inhibition by high levels of fructose, while FKII shows Michaelis-Menten kinetics with fru concentrations up to 10mM (Fig. 3a). The yeast-expressed LeFRK1-Y similarly shows Michaelis-Menten kinetics with fru concentrations up to 10mM, while LeFRK2-Y shows the characteristic of substrate inhibition by high levels of fructose (Fig. 3b).
Fig. 3. Effect of fru concentration on the activity of the three fructokinase isoforms from (A) immature fruit pericarp and from (B) yeastexpressed LeFRK genes. Data are expressed as percentage of maximal activity for each enzyme in order to emphasize the similar and distinct patterns of activity. Maximal activities were, nmol g 1 fw min 1: FK1, 710; FKII, 38; FKIII, 71; LeFRK1-Y, 148; LeFRK2-Y, 238.
2.2.2. Inhibition by Mg Of the fruit enzymes only FKI shows the characteristic of inhibition by Mg concentrations above 1 mM (Fig. 4a). FKII shows no inhibition by Mg while FKIII shows little, if any, inhibition. Of the yeast-expressed enzymes only LeFRK2-Y is inhibited by Mg concentrations above 1 mM, while LeFRK1-Y is not (Fig. 4b). 2.2.3. Nucleotide specificity The three fruit enzymes responded differently to the four nucleotide substrates (Fig. 5 a–c). FKI had highest activity with ATP and GTP at concentrations up to 0.5 mM. However, above 0.5 mM this enzyme showed inhibition by GTP and CTP. FKII and FKIII showed preferred activity with ATP with no indication of inhibition by high NTP concentrations. All three enzymes also showed product inhibition by ADP (not shown). The two yeast-expressed enzymes also responded differently to the four nucleotide substrates (Fig. 6a and b). LeFRK2-Y responded in a similar fashion as the tomato fruit FKI, with highest activity with ATP and GTP at concentrations up to 0.5 mM, together with inhibition by GTP and CTP at concentrations above 0.5 mM. LeFRK1-Y showed preferred activity with ATP alone. Both enzymes also showed product inhibition by ADP.
Fig. 4. Effect of Mg concentration on the activity of the three fructokinase isoforms from (A) immature fruit pericarp and from (B) yeastexpressed LeFRK genes. Data are expressed as percentage of maximal activity for each enzyme. Maximal activities were, in nmol ml 1 min 1: FK1, 840; FKII, 52; FKIII, 161; LeFRK1-Y, 150; LeFRK2-Y, 226.
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Fig. 6. Effect of nucleotide concentrations on the activity of the two yeast expressed fructokinase enzymes. (A), LeFRKI; (B), LeFRKII.
Fig. 5. Effect of nucleotide concentrations on the activity of the three fructokinase isoforms from immature tomato fruit pericarp. (A), FKI; (B), FKII; (C) FKIII.
2.2.4. Coelution of yeast-expressed and tomato fruit fructokinase Each of the LeFkr-Y gene products were chromatographed individually, and also together with the enzyme extract from tomato pericarp, in order to determine whether they coelute with any of the tomato enzymes. LeFRK2-Y coeluted with the tomato FKI under the same conditions used for the separation of the fruit enzymes. The LeFRK1-Y protein eluted slightly after FKII, coeluting with the second hexokinase enzyme, HK2 (not shown).
3. Discussion We report here three distinct forms of fructokinase in tomato fruit, which can be distinguished one from the other on the basis of their responses to fructose, Mg and NTP concentrations. The first enzyme which eluted from ion-exchange chromatography, FKI, was inhibited by both high fru and high Mg. It also showed a characteristic and unique response to NTP concentration,
with inhibition at high concentrations of GTP and CTP. The second eluted enzyme, FKII, had lowest affinity for fru and was inhibited by neither fru nor Mg, while the third enzyme, FKIII, was inhibited by fru but not by Mg. These distinguishing characterisitics allowed us to compare the gene products for the two known LeFRK genes and to attempt to assign gene–product relationships between the genes and the native enzymes. The LeFRK2 gene encodes for the FK1 protein, based on the similarities in fru inhibition, Mg inhibition and, especially, the unique response to NTPs, together with the coelution of the two proteins. To further prove this identity we have recently shown that antisense FRK2 tomato fruit show a complete knockout of the FKI protein specifically, while the other two peaks of activity are unmodified (unpublished). The gene product of LeFRK1 appears likely to be the FKII protein, based on their similar low affinity for fructose and the absence of inhibition by either fru or Mg. Although the LeFRK1-Y protein product eluted slightly after the native FKII protein, this may be due to different post-translational modifications in plants and yeast, as previously suggested (Dai et al., 1997; Veramendi et al., 1999). The FKIII protein may be either be a distinct gene product or a post-translationally modified product of either of the two known genes. However, considering the distinguishing characterisitics of fru and Mg inhibition, together with the large difference in ionic strength required to elute the protein, it is reasonable to suggest that it is, in fact, a product of a third gene. Pego and Smeekens (2000) reported a third FK
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gene in Arabidopsis and we have found an additional FK gene in tomato which is exclusively expressed in anthers, and is presently under study (unpublished). Multiple fructokinases have been reported in other plant tissues. In potato tuber three enzymes were separated by ion-exchange chromatography (Gardner et al., 1992; Renz and Stitt, 1993) but in one study all three forms were inhibited by high fructose concentrations (Renz and Stitt, 1993) while in the other report only the first two eluted proteins were inhibited (Gardner et al., 1992). Similarly, Martinez-Barajas and Randall (1996) reported two tomato fruit isoforms and considering their relatively similar kinetic characterisitics they are possibly due to minor modifications of the LeFRK2 gene product (Martinez-Barajas et al., 1997). Regretfully, much of the enzyme data for FKs available in the literature cannot be directly compared, due to the different techniques used in separation and enzyme characterization. Pego and Smeekens (2000) recently attempted to clarify the confusion regarding the different fructokinases in plants and suggested that the reported fructokinase isoforms may be attributed to only two different enzymes. Considering that so few genes for fructokinases have so far been characterized, the classification of the fructokinases will undoubtedly become clearer with additional gene cloning, which will allow for classification based on homologies between a large population of sequences from numerous plant species. Fructokinase activity in tomato fruit has been associated with the transient accumulation of starch (Schaffer and Petreikov, 1997a). Kanayama et al. (1997, 1998) showed that the temporal and spatial expression of the LeFRK2 gene in tomato fruit coincides with, although is not limited to, this transient starch accumulation and suggested that the LeFRK2 gene product was particularly important for starch synthesis. We therefore hypothesized that since the FKI protein is the LeFRK2 gene product, its activity would most likely be absent or relatively low, in comparison to the other FK enzymes in the mature fruit. The results of the present study show that the LeFRK2 gene product remains the major native FK in the ripe fruit. It does not undergo any particular loss of activity, compared to the other fructokinases and its suggested specific role in transient starch synthesis is not certain. The distinguishing characteristics of the three enzymes (inhibition by fru and Mg and response to nucleotides) may be of physiological significance, perhaps related to distinct localization and compartmentation of the isoforms. The trait of fructose inhibition has been proposed as a possible classifying characterisitc of fructokinase enzymes (Pego and Smeekens, 2000) and its physiological significance may be related to a parallel fructose inhibition of the sucrose synthase enzyme at physiological levels of fructose found in tomato fruit (Schaffer and Petreikov, 1997b) leading to a ‘double
brake’ mechanism which may control sucrose metabolism flux (Kanayama et al., 1997, 1998; Schaffer and Petreikov, 1997b; Pego and Smeekens, 2000). Based on the similar characteristic of the yeast-expressed enzyme, the trait of Mg inhibition appears to be inherent to the enzyme and not artifactual, although we do not posit physiological significance. Similarly the unique trait of CTP and GTP inhibition of the FKI enzyme is also characteristic of the protein and not artifactual since the heterologous yeast-expressed protein has the identical trait. This study paves the way for the analysis of the physiological significance of the individual tomato fruit fructokinases using plants with genetically modified activities of the individual fructokinases. These studies are in progress.
4. Experimental 4.1. Plant materials and yeast strains Plants of tomato (Lycopersicon esculentum Mill (cv. 7844 and F144) were grown under standard conditions in a greenhouse in Bet Dagan, Israel. Flowers were allowed to self-pollinate. Chemicals and enzymes were purchased from Sigma or Boehringer Mannheim. Yeast strains and transformation methods were as described previously (Kanayama et al., 1998). 4.2. Enzyme extractions and assays Fructokinase (EC 2.7.1.4) and hexokinase (EC 2.7.1.1) activities were extracted from pericarp tissue (4.5 g) of immature green (approximately 15 days after flowering) or red tomato fruit as described in Schaffer and Petreikov (1997a) with the addition of 1 mM PMSF to the extraction buffer. The 25–80% ammonium sulfate precipitate was collected, resuspended in extraction buffer, desalted on Sephadex G-25 and filtered through a 0.2 mm polysulfone membrane (Whatman International Ltd., Maidstone, England). The protein separation was performed on a Shimadzu HPLC system. For the separation of the fructokinase enzymes in young and mature fruit, as well as the yeast-expressed enzymes, the protein extract was applied (flow rate 0.5 ml/min) to a Mono-Q column HR 5/5 (Pharmacia Biotech AB, Uppsala, Sweden) prequilibrated with buffer containing 20 mM HEPES (pH 7.0) and 2.5 mM DTT. Unbound protein was eluted with the same buffer, followed by a 0–0.5 M KCl salt gradient. Protein was monitored at 280 nm. Fractions of 0.5 ml were collected and both fructose and glucose phosphorylating activities were measured. The fractions containing peak activities of the individual isoenzymes were bulked for further characterization.
M. Petreikov et al. / Phytochemistry 58 (2001) 841–847
The yeast-expressed enzymes were extracted as described previously (Kanayama et al., 1998). Enzyme extracts were separated on Mono-Q ion-exchange chromatography under identical conditions as for the fruit pericarp enzyme extracts. For the results presented in Fig. 1 a Mono-P column was used under identical elution and buffer conditions as described for the Mono-Q column. Two different assays were used for measuring fructokinase activity. Assay 1 was conducted with low fructose and magnesium concentrations and the reaction mixture (1 ml) contained 30 mM HEPES (pH 7.6), 9 mM KCl, 0.5 mM MgCl2, 1 mM ATP, 1 mM NAD, 1 unit PGI (type III), 1 unit NAD-dependent G-6-P DH (from Leuconostoc) and the reaction was initiated with 1 mM fructose. Assay 2 for fructokinase activity was conducted with high fructose (10 mM) and magnesium (3 mM) concentrations. For determining hexokinase activity glucose (10 mM) served as substrate, magnesium levels were 3 mM MgCl2 and PGI was excluded from the reaction mixture. All the enzymatic reactions were carried out at 37 C and A340 nm was monitored continuously, as previously described (Petreikov and Schaffer, 1997a). Optimum conditions for each isoenzyme were used in enzyme assays for characterization of the three FK isoforms. For FKI assay 1 (low fru and Mg), while for FKII and FKIII, assay 2 (high fru and M) were used. ADP inhibition studies were carried out using the same enzyme extract with ADP concentrations ranging up to 5.0 mM.
Acknowledgements The assistance of Dr. Lena Yeselson and Shmuel Shen is gratefully acknowledged. References Dai, N., Schaffer, A., Petreikov, M., Granot, D., 1997. Potato (Solanum tuberosum L. ) fructokinase expressed in yeast exhibits inhibition by fructose of both in vitro enzyme activity and rate of cell proliferation. Plant Sci. 128, 191–197.
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Copeland, L., Morell, M., 1985. Hexose kinases from the plant cytosolic fraction of soybean nodules. Plant Physiol. 79, 114–117. Gardner, A., Davies, H.V., Burch, L.R., 1992. Purification and properties of fructokinase from developing tubers of potato (Solanum tuberosum L. ). Plant Physiol. 100, 178–183. Huber, S.C., Akazawa, T., 1985. A novel sucrose synthase pathway for sucrose degradation in cultured sycamore cells. Plant Physiol 81, 1008–1011. Kanayama, Y., Dai, N., Granot, D., Petreikov, M., Schaffer, A., Bennett, A.B., 1997. Divergent fructokinase genes are differnetially expressed in tomato (Lycopersicon esculentum Mill. ). Plant Physiol. 113, 1379–1384. Kanayama, Y., Granot, D., Dai, N., Petreikov, M., Schaffer, A., Powell, A., Bennett, A.B., 1998. Tomato fructokinases exhibit differential expression and substrate regulation. Plant Physiol. 117, 85–90. Martinez-Barajas, E., Randall, D.D., 1996. Purification and characterization of fructokinase from developing tomato (Lycopersicon esculentum Mill.) fruits. Planta 199, 451–458. Martinez-Barajas, E., Luethy, M.H., Randall, D.D., 1997. Molecular cloning and analysis of fructokinase exoression in tomato (Lycopersicon esculentum Mill.). Plant Sci. 125, 13–20. Nakamura, N., Shimizu, M., Suzuki, H., 1991. Characterization of hexose kinases from camellia and lily pollen grains. Physiol. Plant. 81, 215–220. Renz, A., Merlo, L., Stitt, M., 1993. Partial purification from potato tubers of three fructokinases and three hexokinases which show different organ and developmental specificity. Planta 190, 156–165. Renz, A., Stitt, M., 1993. Substrate specificity and product inhibition of different forms of fructokinases and hexokinases in developing potato tubers. Planta 190, 166–175. Ruan, Y.-L., Patrick, J.W., 1995. The cellular pathway of postphloem sugar transport in developing tomato fruit. Planta 196, 434–444. Schaffer, A.A., Petreikov, M., 1997a. Sucrose-to-starch metabolism in tomato fruit undergoing transient starch accumulation. Plant Physiol. 113, 739–746. Schaffer, A.A., Petreikov, M., 1997b. Inhibition of fructokinase and sucrose synthase by physiological levels of fructose in young tomato fruit undergoing transient starch accumulation. Physiol. Plant. 110, 800–806. Turner, J.F., Garrison, D.D., Copeland, L., 1977. Fructokinase (fraction IV) of pea seeds. Plant Physiol. 60, 666–669. Veramendi, J., Roessner, U., Renz, A., Willmitzer, L., Trethwey, R.N., 1999. Antisense repression of hexokinase 1 leads to an overaccumulation of starch in leaves of transgenic potato plants but not to significant changes in tuber carbolydrate metabolism. Plant Physiol. 121, 123–133. Viola, R., 1996. Hexose metabolism in discs excised from developing potato (Solanum tuberosum) tubers. II. Estimations of fluxes in vivo and evidence that fructokinase catalyses a near rate-limiting reaction. Planta 198, 186–196.
Characterization of native and yeast-expressed tomato fruit fructokinase enzymes Marina Petreikov, Nir Dai, David Granot, Arthur A. Schaffer* Department of Vegetable Crops, Agricultural Research Organization, Volcani Center, Bet Dagan, 50250, Israel Received 8 April 2001; received in revised form 16 July 2001
Abstract Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Lycopersicon esculentum Mill.; Hexokinase; Substrate inhibition; Gene product
1. Introduction The developing tomato fruit sink metabolizes translocated suc via either invertase or suc synthase. The path of sucrose catabolism is developmentally regulated: the sucrose synthase pathway is associated with the young fruit which transiently accumulates starch, while the invertase pathway is associated with the later stages of fruit development (Ruan and Patrick, 1995; Ho, 1996; Schaffer and Petreikov, 1997a). In addition to changes in the biochemical pathway, the transition between the two developmental stages is accompanied by changes in the translocation pathway, from a symplastic to an apoplastic one, as well (Ruan and Patrick, 1995; Ho, 1996). Irrespective of the initial enzyme in the pathway, free fru accounts for one half of the imported suc and its phosphorylation is requisite for its further metabolism. Fru phosphorylation may be carried out either by * Corresponding author. Tel.: +972-3-9683646; fax: +972-39669642. E-mail address: [email protected] (A.A. Schaffer).
hexokinases (EC 2.7.1.1), which can phosphorylate both glucose and fructose, or by specific fructokinases (EC 2.7.1.4). The irreversible fructokinase reaction is likely to play an important role in determining carbon flux in sink tissue (Viola, 1996). The activity pattern of fructokinase in developing tomato fruit, but not hexokinase, parallels the transient starch content in the fruit (Schaffer and Petreikov, 1997a). Fructokinases have been characterized from a number of plant tissues (see recent review by Pego and Smeekens, 2000). It is not uncommon to find two or three isoforms upon separation by ion-exchange chromatography (i.e. potato tuber, Gardner et al., 1992; Renz and Stitt 1993) which may also show tissue and developmental specifity (Renz et al., 1993). Some fructokinases show substrate inhibition by fructose (i.e. tomato, Schaffer and Petreikov, 1997b; potato, Gardner et al. 1992; Renz and Stitt, 1993) which may be physiologically significant in the case of tomato fruit which contains high fructose levels (Schaffer and Petreikov, 1997b). Fructokinases can also be characterized by their nucleotide specificity; Huber and Akazawa (1985) reported a fructokinase utilizing UTP while most other
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00331-4
842
M. Petreikov et al. / Phytochemistry 58 (2001) 841–847
reported fructokinases show relative specificity to ATP. FKs can also be distinguished by the characteristic of inhibition by excess Mg ions. The fructokinase from pollen grains (Nakamura et al., 1991) and from soybean nodules (Copeland and Morell, 1985) was inhibited by Mg concentrations above 1–2 mM while that of pea seeds was not (Turner et al., 1977). A fructokinase from tomato fruit similarly showed the phenomenon of Mg inhibition (Schaffer and Petreikov, 1997b). Tomato fruit fructokinases have recently attracted increasing attention due to their possible role in transient starch accumulation (Schaffer and Petreikov, 1997a; Kanayama et al., 1997, 1998), in addition to possible physiological roles in hexose accumulation and sugar sensing (Pego and Smeekens, 2000). MartinezBarajas and Randall (1996) separated two isoforms of fructokinase from tomato fruit, which showed similar sensitivity to inhibition by fructose and similar nucleotide specificity. However, Kanayama et al. (1997) cloned two fructokinase genes from tomato with different expression patterns, as well as different kinetic characteristics of the expressed protein. One of the genes (LeFRK2) which is expressed primarily in the early stages of fruit development yields a gene product which, when expressed in yeast shows inhibition by fructose (Kanayama et al., 1998). The other gene (LeFRK1) is expressed throughout fruit development (Kanayama et al 1997) and its yeast-expressed gene product is not inhibited by fructose (Kanayama et al., 1998). The LeFRK2 gene was hypothesized to be responsible for the fructokinase activity associated with transient starch accumulation (Kanayama et al., 1998) while the LeFRK1 gene was suggested to play a housekeeping role in carbohydrate metabolism throughout the plant, supplying fru6P for glycolysis. In light of these recent data we reinvestigated fructokinase activity in developing tomato fruit. We report here three FK isoforms, which can be distinguished one from the other on the basis of fru and Mg inhibition and nucleotide specificity. Furthermore, we attempt to relate the two known LeFRK gene products to the FK isoforms present in the tomato fruit.
observed using two assay methods. One assay contained low fru (1.0 mM) and low Mg (0.5 mM), with which the FKI enzyme showed maximal activity. The second assay contained high fru (10 mM) and high Mg (3 mM), which gave maximal activities for FKII. The FKIII enzyme, which eluted only under high ionic concentration, showed approximately equal activities with both assays. 2.1.2. Activity during development The total FK activity in crude extracts of immature (15 DAA) and ripe fruit pericarp is presented in Table 1. Recovery experiments indicated that differences in extractable activity between the different stage fruit were not artifactually due to inhibitory substances in the mature fruit. Similar recovery of a known amount of purified FKI (from the MonoQ fractions, as described further on) was obtained from both fruit developmental stages (not shown). Activity with both assay systems is higher in the young fruit and sharply declines in the more mature fruit. At both stages of fruit development activity is higher with the low fru/low Mg assay system, indicating that even in the mature fruit the major activity is inhibited by excess fructose and Mg. A comparison of the chromatograms of the pericarp fructokinase enzymes from immature (15 DAA) and ripe fruit stages (Fig. 2) indicates that the three peaks of fructose phosphorylation activity were observed in both stages. The enzyme first eluted (FKI), assayed under low substrate conditions, remained the dominant peak of activity even in the ripe fruit. The FKII enzyme was present in the ripe fruit but generally was the least active of the three FKs. 2.2. Characterization of the three tomato fruit FK enzymes and comparison with the two yeast-expressed LeFRK (LeFRK-Y) gene products In order to determine the relationships between the native FK enzymes and the products of the two LeFRK genes, enzyme extracts from mutant yeast lacking endogenous hexose kinase activity and expressing each of the two described LeFRK genes were separated on
2. Results 2.1. Fructokinase isoenzymes during fruit development
Table 1 Fructokinase activity in immature and mature tomato fruit pericarp Maturity stage
2.1.1. Separation on HPLC-ion exchange Hexose phosphorylation activity from young tomato fruit were separated by HPLC-ion exchange chromatography (Fig. 1). We observed three peaks of activity with fructose as substrate, as well as two additional peaks of activity with glucose (termed HKI and HKII). The three fructose phosphorylating peaks (termed FKI, FKII and FKIII, according to the order of elution) were
Fructokinase activity (nmol g
1
fw min 1)
Assay 1a
Assay 2b
Immature
184+25
85+8
Mature
63+8
27+1
a
Assay 1 contained low fru (1.0 mM) and low Mg (0.5 mM). Assay 2 contained high fru (10 mM) and high Mg (3 mM). Data are averages+S.E. of 4 (immature) and 7 (mature) separate extractions from individual fruit. b
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Fig. 1. HPLC-ion exchange (MonoP) separation of hexose kinase activity in immature tomato fruit pericarp. Assay 1 contained low fru (1.0 mM) and low Mg (0.5 mM). Assay 2 contained high fru or glu (10 mM) and high Mg (3 mM).
Fig. 2. HPLC-ion exchange (MonoQ) separation of fructokinase activity in immature (A) and mature (B) tomato fruit pericarp. Assays are as in Fig. 1. Note the different scales in the two chromatograms.
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MonoQ ion chromatography under the same conditions as the tomato extract (not shown). The partially purified native FK enzymes and the yeast-expressed enzymes were partially characterized and compared as described below. 2.2.1. Hexose specificity and inhibition by fru All three native fruit enzymes were specific for fru, with no discernible activity with glu or mannose (not shown). Of the three enzymes FKII had the lowest affinity for the substrate (Fig. 3a). Both yeast-expressed enzymes were also specific for fru, with no discernible activity with glucose or mannose. The LeFRK1-Y enzyme has an affinity pattern for fru analogous to FKII (Fig. 3b). Both FKI and FKIII from tomato fruit show the characteristic of substrate inhibition by high levels of fructose, while FKII shows Michaelis-Menten kinetics with fru concentrations up to 10mM (Fig. 3a). The yeast-expressed LeFRK1-Y similarly shows Michaelis-Menten kinetics with fru concentrations up to 10mM, while LeFRK2-Y shows the characteristic of substrate inhibition by high levels of fructose (Fig. 3b).
Fig. 3. Effect of fru concentration on the activity of the three fructokinase isoforms from (A) immature fruit pericarp and from (B) yeastexpressed LeFRK genes. Data are expressed as percentage of maximal activity for each enzyme in order to emphasize the similar and distinct patterns of activity. Maximal activities were, nmol g 1 fw min 1: FK1, 710; FKII, 38; FKIII, 71; LeFRK1-Y, 148; LeFRK2-Y, 238.
2.2.2. Inhibition by Mg Of the fruit enzymes only FKI shows the characteristic of inhibition by Mg concentrations above 1 mM (Fig. 4a). FKII shows no inhibition by Mg while FKIII shows little, if any, inhibition. Of the yeast-expressed enzymes only LeFRK2-Y is inhibited by Mg concentrations above 1 mM, while LeFRK1-Y is not (Fig. 4b). 2.2.3. Nucleotide specificity The three fruit enzymes responded differently to the four nucleotide substrates (Fig. 5 a–c). FKI had highest activity with ATP and GTP at concentrations up to 0.5 mM. However, above 0.5 mM this enzyme showed inhibition by GTP and CTP. FKII and FKIII showed preferred activity with ATP with no indication of inhibition by high NTP concentrations. All three enzymes also showed product inhibition by ADP (not shown). The two yeast-expressed enzymes also responded differently to the four nucleotide substrates (Fig. 6a and b). LeFRK2-Y responded in a similar fashion as the tomato fruit FKI, with highest activity with ATP and GTP at concentrations up to 0.5 mM, together with inhibition by GTP and CTP at concentrations above 0.5 mM. LeFRK1-Y showed preferred activity with ATP alone. Both enzymes also showed product inhibition by ADP.
Fig. 4. Effect of Mg concentration on the activity of the three fructokinase isoforms from (A) immature fruit pericarp and from (B) yeastexpressed LeFRK genes. Data are expressed as percentage of maximal activity for each enzyme. Maximal activities were, in nmol ml 1 min 1: FK1, 840; FKII, 52; FKIII, 161; LeFRK1-Y, 150; LeFRK2-Y, 226.
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Fig. 6. Effect of nucleotide concentrations on the activity of the two yeast expressed fructokinase enzymes. (A), LeFRKI; (B), LeFRKII.
Fig. 5. Effect of nucleotide concentrations on the activity of the three fructokinase isoforms from immature tomato fruit pericarp. (A), FKI; (B), FKII; (C) FKIII.
2.2.4. Coelution of yeast-expressed and tomato fruit fructokinase Each of the LeFkr-Y gene products were chromatographed individually, and also together with the enzyme extract from tomato pericarp, in order to determine whether they coelute with any of the tomato enzymes. LeFRK2-Y coeluted with the tomato FKI under the same conditions used for the separation of the fruit enzymes. The LeFRK1-Y protein eluted slightly after FKII, coeluting with the second hexokinase enzyme, HK2 (not shown).
3. Discussion We report here three distinct forms of fructokinase in tomato fruit, which can be distinguished one from the other on the basis of their responses to fructose, Mg and NTP concentrations. The first enzyme which eluted from ion-exchange chromatography, FKI, was inhibited by both high fru and high Mg. It also showed a characteristic and unique response to NTP concentration,
with inhibition at high concentrations of GTP and CTP. The second eluted enzyme, FKII, had lowest affinity for fru and was inhibited by neither fru nor Mg, while the third enzyme, FKIII, was inhibited by fru but not by Mg. These distinguishing characterisitics allowed us to compare the gene products for the two known LeFRK genes and to attempt to assign gene–product relationships between the genes and the native enzymes. The LeFRK2 gene encodes for the FK1 protein, based on the similarities in fru inhibition, Mg inhibition and, especially, the unique response to NTPs, together with the coelution of the two proteins. To further prove this identity we have recently shown that antisense FRK2 tomato fruit show a complete knockout of the FKI protein specifically, while the other two peaks of activity are unmodified (unpublished). The gene product of LeFRK1 appears likely to be the FKII protein, based on their similar low affinity for fructose and the absence of inhibition by either fru or Mg. Although the LeFRK1-Y protein product eluted slightly after the native FKII protein, this may be due to different post-translational modifications in plants and yeast, as previously suggested (Dai et al., 1997; Veramendi et al., 1999). The FKIII protein may be either be a distinct gene product or a post-translationally modified product of either of the two known genes. However, considering the distinguishing characterisitics of fru and Mg inhibition, together with the large difference in ionic strength required to elute the protein, it is reasonable to suggest that it is, in fact, a product of a third gene. Pego and Smeekens (2000) reported a third FK
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gene in Arabidopsis and we have found an additional FK gene in tomato which is exclusively expressed in anthers, and is presently under study (unpublished). Multiple fructokinases have been reported in other plant tissues. In potato tuber three enzymes were separated by ion-exchange chromatography (Gardner et al., 1992; Renz and Stitt, 1993) but in one study all three forms were inhibited by high fructose concentrations (Renz and Stitt, 1993) while in the other report only the first two eluted proteins were inhibited (Gardner et al., 1992). Similarly, Martinez-Barajas and Randall (1996) reported two tomato fruit isoforms and considering their relatively similar kinetic characterisitics they are possibly due to minor modifications of the LeFRK2 gene product (Martinez-Barajas et al., 1997). Regretfully, much of the enzyme data for FKs available in the literature cannot be directly compared, due to the different techniques used in separation and enzyme characterization. Pego and Smeekens (2000) recently attempted to clarify the confusion regarding the different fructokinases in plants and suggested that the reported fructokinase isoforms may be attributed to only two different enzymes. Considering that so few genes for fructokinases have so far been characterized, the classification of the fructokinases will undoubtedly become clearer with additional gene cloning, which will allow for classification based on homologies between a large population of sequences from numerous plant species. Fructokinase activity in tomato fruit has been associated with the transient accumulation of starch (Schaffer and Petreikov, 1997a). Kanayama et al. (1997, 1998) showed that the temporal and spatial expression of the LeFRK2 gene in tomato fruit coincides with, although is not limited to, this transient starch accumulation and suggested that the LeFRK2 gene product was particularly important for starch synthesis. We therefore hypothesized that since the FKI protein is the LeFRK2 gene product, its activity would most likely be absent or relatively low, in comparison to the other FK enzymes in the mature fruit. The results of the present study show that the LeFRK2 gene product remains the major native FK in the ripe fruit. It does not undergo any particular loss of activity, compared to the other fructokinases and its suggested specific role in transient starch synthesis is not certain. The distinguishing characteristics of the three enzymes (inhibition by fru and Mg and response to nucleotides) may be of physiological significance, perhaps related to distinct localization and compartmentation of the isoforms. The trait of fructose inhibition has been proposed as a possible classifying characterisitc of fructokinase enzymes (Pego and Smeekens, 2000) and its physiological significance may be related to a parallel fructose inhibition of the sucrose synthase enzyme at physiological levels of fructose found in tomato fruit (Schaffer and Petreikov, 1997b) leading to a ‘double
brake’ mechanism which may control sucrose metabolism flux (Kanayama et al., 1997, 1998; Schaffer and Petreikov, 1997b; Pego and Smeekens, 2000). Based on the similar characteristic of the yeast-expressed enzyme, the trait of Mg inhibition appears to be inherent to the enzyme and not artifactual, although we do not posit physiological significance. Similarly the unique trait of CTP and GTP inhibition of the FKI enzyme is also characteristic of the protein and not artifactual since the heterologous yeast-expressed protein has the identical trait. This study paves the way for the analysis of the physiological significance of the individual tomato fruit fructokinases using plants with genetically modified activities of the individual fructokinases. These studies are in progress.
4. Experimental 4.1. Plant materials and yeast strains Plants of tomato (Lycopersicon esculentum Mill (cv. 7844 and F144) were grown under standard conditions in a greenhouse in Bet Dagan, Israel. Flowers were allowed to self-pollinate. Chemicals and enzymes were purchased from Sigma or Boehringer Mannheim. Yeast strains and transformation methods were as described previously (Kanayama et al., 1998). 4.2. Enzyme extractions and assays Fructokinase (EC 2.7.1.4) and hexokinase (EC 2.7.1.1) activities were extracted from pericarp tissue (4.5 g) of immature green (approximately 15 days after flowering) or red tomato fruit as described in Schaffer and Petreikov (1997a) with the addition of 1 mM PMSF to the extraction buffer. The 25–80% ammonium sulfate precipitate was collected, resuspended in extraction buffer, desalted on Sephadex G-25 and filtered through a 0.2 mm polysulfone membrane (Whatman International Ltd., Maidstone, England). The protein separation was performed on a Shimadzu HPLC system. For the separation of the fructokinase enzymes in young and mature fruit, as well as the yeast-expressed enzymes, the protein extract was applied (flow rate 0.5 ml/min) to a Mono-Q column HR 5/5 (Pharmacia Biotech AB, Uppsala, Sweden) prequilibrated with buffer containing 20 mM HEPES (pH 7.0) and 2.5 mM DTT. Unbound protein was eluted with the same buffer, followed by a 0–0.5 M KCl salt gradient. Protein was monitored at 280 nm. Fractions of 0.5 ml were collected and both fructose and glucose phosphorylating activities were measured. The fractions containing peak activities of the individual isoenzymes were bulked for further characterization.
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The yeast-expressed enzymes were extracted as described previously (Kanayama et al., 1998). Enzyme extracts were separated on Mono-Q ion-exchange chromatography under identical conditions as for the fruit pericarp enzyme extracts. For the results presented in Fig. 1 a Mono-P column was used under identical elution and buffer conditions as described for the Mono-Q column. Two different assays were used for measuring fructokinase activity. Assay 1 was conducted with low fructose and magnesium concentrations and the reaction mixture (1 ml) contained 30 mM HEPES (pH 7.6), 9 mM KCl, 0.5 mM MgCl2, 1 mM ATP, 1 mM NAD, 1 unit PGI (type III), 1 unit NAD-dependent G-6-P DH (from Leuconostoc) and the reaction was initiated with 1 mM fructose. Assay 2 for fructokinase activity was conducted with high fructose (10 mM) and magnesium (3 mM) concentrations. For determining hexokinase activity glucose (10 mM) served as substrate, magnesium levels were 3 mM MgCl2 and PGI was excluded from the reaction mixture. All the enzymatic reactions were carried out at 37 C and A340 nm was monitored continuously, as previously described (Petreikov and Schaffer, 1997a). Optimum conditions for each isoenzyme were used in enzyme assays for characterization of the three FK isoforms. For FKI assay 1 (low fru and Mg), while for FKII and FKIII, assay 2 (high fru and M) were used. ADP inhibition studies were carried out using the same enzyme extract with ADP concentrations ranging up to 5.0 mM.
Acknowledgements The assistance of Dr. Lena Yeselson and Shmuel Shen is gratefully acknowledged. References Dai, N., Schaffer, A., Petreikov, M., Granot, D., 1997. Potato (Solanum tuberosum L. ) fructokinase expressed in yeast exhibits inhibition by fructose of both in vitro enzyme activity and rate of cell proliferation. Plant Sci. 128, 191–197.
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