Vector construction and plant transformation.

Two different modified rat liver bifunctional enzyme cDNAs coding a polypeptide with fru 2,6P2ase activity (Li et al., 1992) and a polypeptide with only 6PF2K activity (Kurland et al., 1992; Tauler et al., 1990) were used. These sequences were inserted via subcloning steps into pBIN19 under the control of CaMV 35S promoter and introduced into Agrobacterium tumefaciens strain LBA 4404 (Scott et al., 1995, 2000). Carnation leaves were transformed as described by Van Altvorst et al. (1995).

Plant material and growth conditions.

Carnation (D. caryophyllus, L.) variety Improved White Sim was obtained from the Óbuda Horticultural Laboratory (Budapest, Hungary). Transgenic shoots were potted in soil and grown in a climate chamber in a 16-h light/8-h dark regime with 200 W·m⁻² light intensity at 20 ºC. Leaves were harvested at different time intervals from the start of the illumination period (0, 1, 2, 4, 6, and 8 h). The plants were sampled 3 months after potting.

Measurement of enzyme activities and metabolites.

Enzyme extraction and activity assays were performed as described by Hatzfeld et al. (1990), Scott et al. (1995), Sweetlove et al. (1996) and Veramendi et al. (2002). Fructose 2,6-bisphosphate was extracted with NaOH according to the method of Ball and Rees (1988) and assayed by the method of Van Schaftingen et al. (1982).

Extraction and measurement of sucrose, starch, glucose, and fructose were performed by using Boehringer Mannheim test combination kits according to the manufacturer's instructions. Phosphorylated intermediates were assayed as described by Michal (1984a, b).

Statistical analysis.

The Student t test was performed using Microsoft Office Excel 2003 (Microsoft Corporation, Seattle). Significant differences between two sets of data were assessed at P < 0.05.

Fructose 2,6-bisphosphate content.

To investigate the effect of altered fru 2,6P₂ content on carbohydrate metabolism, transgenic carnation harboring two types of modified rat liver bifunctional enzyme cDNAs (6PF2K/fru 2,6P2ase) was produced (Szőke et al., 2006). The most substantial difference in the amounts of fru 2,6P₂ was experienced 4 h after the start of illumination (data not shown). Therefore, the samples were taken from plants at this time of the photoperiod to assess the consequences of altered fru 2,6P₂ contents on enzyme activities and to determine how these changes in enzyme activities affect the important carbohydrate and phosphorylated intermediate concentrations. Four independent transgenic lines (F406 and F407, and P207 and P228) were characterized in our experiment. In plants transformed with the phosphatase construct (F406 and F407), the fru 2,6P₂ levels decreased from 45% to 70% compared with the wild type. Conversely, transformation with the kinase construct (P207 and P228) caused a 45% to 85% increase in fru 2,6P₂ content (Table 1). In comparison with transgenic tobacco plants, expressing the same genes of rat liver origin with only phosphatase activity, the fru 2,6P₂ content decreased by 54% to 75% (Scott et al., 2000). At the same time, tobacco plants harboring the gene with kinase activity alone contained 130% and 214% more fru 2,6P₂ than the wild-type plants (Scott et al., 1995). This increase in Kalanchoe ranged from 228% to 350% (Truesdale et al., 1999). More efficient reduction in fru 2,6P₂ levels was obtained in Arabidopsis (up to 5% of wild type) and potato (up to 10% of wild type) transformed with an endogenous bifunctional enzyme gene in sense or antisense orientation (Draborg et al., 2001; Rung et al., 2004).

Table 1. Effect of fructose 2,6-bisphosphate content on enzyme activities in leaves of wild-type and transgenic plant lines (F406 and F407, plants harboring phosphatase construct; P207 and P228, plants transformed with kinase construct).

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Enzyme activities.

There were significant changes in enzyme activities influenced by fru 2,6P₂ content (Table 1). Elevated fru 2,6P₂ levels inhibited the activity of FBPase and stimulated that of PFP. Fructose 1,6-bisphophatase catalyzes the hydrolysis of fru 1,6P₂ to fructose 6-phosphate (fru6P), and it is an essential enzyme in sucrose biosynthesis. Pyrophosphate:fructose 6-phosphate-1-phosphotransferase has a different role to play. It determines the rate-limiting step of glycolysis and gluconeogenesis, the reversible conversion between fru 1,6P₂ and fru6P. The direction of the reaction depends on the concentration of substrates and products (Stitt, 1990). The decreased fru 2,6P₂ concentrations resulted in higher FBPase activities in both transgenic lines. The PFP activity decreased significantly in the F406 line, but increased in the F407 plant (Table 1). Thus, the effect of the reduced amount of fru 2,6P₂ on PFP activities was not clear. In tobacco and Arabidopsis, reduced fru 2,6P₂ levels did not cause any significant differences in the enzyme activities between transgenic and wild-type plants (Draborg et al., 2001; Scott et al., 2000), but elevated fru 2,6P₂ levels in tobacco affected key enzyme activities (Scott et al., 1995). These variations may reflect differences between plant species. The enzyme 6-phosphofructo-1-kinase (PFK) in nonplant organisms—as a catalyst of the irreversible conversion of fru6P to fru 1,6P₂—was found to be activated by fru 2,6P₂ (Van Schaftingen, 1987). In plants it was determined that PFK is insensitive to fru 2,6P₂ concentrations (Stitt, 1990). On the other hand, decreased fru 2,6P₂ contents lowered PFK activities by 10% to 54% (F406 and F407 plants), at the same time the elevated fru 2,6P₂ levels caused a slight, 8% activation of PFK in the P207 plant, whereas in the P228 line the PFK activity decreased by 11% compared with the wild type (Table 1). This means that there is no unambiguous evidence for the effect of elevated fru 2,6P₂ levels on PFK activity. Hexokinase activities—the catalyst of the phosphorylation of hexoses to hexose monophosphates—were not altered significantly in any transgenic plant lines compared with the wild types (Table 1).

Carbohydrates and phosphorylated intermediates.

Reduced fru 2,6P₂ levels stimulated the activity of FBPase (key enzyme of sucrose synthesis) causing a two- to threefold increase in the amount of sucrose in plants expressing bisphosphatase (F406 and F407; Fig. 2A). Experiments with radioactively labeled ¹⁴CO₂ feeding in transgenic Arabidopsis and tobacco also verified that the decreased fru 2,6P₂ levels favor sucrose synthesis (Draborg et al., 2001; Scott et al., 2000). In tobacco and potato this abundant sucrose during the light period was cleaved and accumulated as glucose and fructose (Rung et al., 2004; Scott et al., 2000). We observed an increase in hexose phosphate [fru6P, glucose 6-phosphate (glu6P)] pools (Fig. 3A, B). The 20% to 38% increase in the amounts of these metabolites is not the result of elevated hexokinase activity. There can be two possible explanations: First, when the reduction in fru 2,6P₂ levels releases the inhibition of FBPase, the accumulation of fru6P and glu6P proceeds (Rung et al., 2004); and second, the superoptimal activation of FBPase raises the export of triose phosphates from the chloroplast to the cytosol (Scott et al., 2000), and this can accelerate the conversion of triose phosphate into hexose phosphate.

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Glucose 6-phosphate is an activator of sucrose phosphate synthase (Doehlert and Huber, 1983). Therefore, it can be assumed that the increased glu6P levels can also cause additional stimulation of sucrose synthesis. In contrast, in transgenic Arabidopsis, the decreased fru 2,6P₂ concentrations did not result in glu6P accumulation, indicating the upregulation of sucrose synthesis (Draborg et al., 2001). In agreement with these data, decreased levels of fru 2,6P₂ led to reduced amounts of 3-phosphoglycerate (3PGA) and dihydroxyacetone phosphate as well (Fig. 3C, D). Declines in triose phosphates may stimulate the export of carbon from the chloroplast through the triose phosphate translocator, thus depleting the substrate for starch synthesis (Scott et al., 2000). Accordingly, starch accumulation was 14% to 37% lower than in the wild-type plants, but this difference was not as dramatic as the sucrose overproduction (Fig. 2B).

As for glucose concentration, similar to our data obtained in carnation (Fig. 2C), a considerable increase was also observed in glucose concentration in transgenic tobacco with decreased amounts of fru 2,6P₂. This accumulation can be explained by the hydrolysis of starch in light. Because decreased levels of triose phosphates restricted the activity of AGPase, plants were not able to recycle these glucose surpluses into starch (Scott et al., 2000). In other transgenic plants, stimulation of sucrose synthesis by the expression of yeast acid invertase caused similar glucose accumulation as well (Sonnewald et al., 1991, 1997). In these cases, the hydrolysis of sucrose was also increased and exceeded the glucose phosphorylation capacity of plants. In contrast with these studies (except the potato), significant fructose overproduction was measured in F406 and F407 carnation transgenic lines (Fig. 2D).

In transgenic plants expressing kinase resulting from elevated fru 2,6P₂ levels (P207 and P228) generally different results were obtained with regard to carbohydrates and phosphorylated intermediate concentrations than in plants expressing bisphosphatase (Fig. 2, 3). In these transgenic plants an increase in the content of triose phosphates was observed (Fig. 3C, D). This could be accompanied by a decrease in the inorganic phosphate (Pi) levels and will result in an increase in the 3PGA-to-Pi ratio (Stitt, 1990), activating AGPase and stimulating starch synthesis in the chloroplast (Scott and Kruger, 1995). In the P207 and P228 lines, AGPase activity was stimulated, which led to a 32% to 40% increase in starch accumulation (Fig. 2B). In transgenic tobacco containing elevated fru 2,6P₂ levels, the higher rate of accumulation of starch was attributed to a decrease in the rate of net starch degradation in the dark period rather than to an increase in starch synthesis during the light period (Scott and Kruger, 1995). We did not examine this possibility in carnation.

The amounts of sucrose, glucose, and hexose phosphates decreased compared with wild-type plants (Figs 2A, C and Fig. 3A, B). Elevated fru 2,6P₂ levels have a direct inhibitory effect on FBPase activity, which prevents the conversion of triose phosphates to hexose phosphates. As a result, sucrose synthesis was restricted to 32% to 40% of that of the wild-type plants. There can be another explanation, too. The increased amounts of fru 2,6P₂ activate PFP, resulting in an enhanced rate of recycling of triose phosphates to hexose phosphates (Fernie et al., 2001). Unexpectedly, the fructose content—similarly to the F406 and F407 transgenic lines—was elevated (Fig. 2D). The reason for this is unknown and requires further investigation.

Knowledge of the role of fru 2,6P₂ in sink tissues is still rather limited. Comparative studies in transgenic potato revealed the different role of fru 2,6P₂ in sink-source tissues. The influence of fru 2,6P₂ in tuber carbohydrate metabolism was less pronounced and had no effect on carbohydrate contents in intact tubers (Rung et al., 2004). In heterotrophic tobacco cells, elevated fru 2,6P₂ levels showed increased rates of cycling of triose phosphates and hexose phosphates (Fernie et al., 2001).

In transgenic plants, bifunctional enzyme genes (6PF2K/fru 2,6P2ase) of different origin were expressed under the control of the constitutive 35S CaMV promoter (Draborg et al., 2001; Rung et al., 2004; Scott et al., 1995, 2000; Truesdale et al., 1999). Additional understanding of the sink source regulation by fru 2,6P₂ and the application of tissue and organ-specific promoters may enable us to enhance the amounts of different carbohydrates in special tissues and organs in agronomically important species.

In summary, in the current study the effects of down-/upregulated fru 2,6P₂ contents on the levels of several enzymes and carbohydrate compounds were described. A decrease in fru 2,6P₂ concentrations favored sucrose synthesis, whereas elevated fru 2,6P₂ contents supported starch accumulation. These results also indicate the key role of fru 2,6P₂ in regulating carbohydrate metabolism of photosynthetic tissues, which is in agreement with the results in transgenic plants presented by Scott et al. (1995, 2000), Draborg et al. (2001), and Rung et al. (2004). Modification of this signal metabolite in carnation provides a powerful tool to modify carbohydrate composition in a complex manner.