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Keywords:

  • Invertase;
  • tomato;
  • soluble solids;
  • sucrose;
  • hexose

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
  • • 
    The effects are shown here of genetic manipulation of invertase on fruit sugar metabolism in tomato.
  • • 
    Introgression of the acid invertase gene on chromosome 3 from Lycopersicon pimpinellifolium (an accession containing high invertase activity) into Lycopersicon esculentum (with relatively low activity) was used to study effects on fruit development and both sugar composition and content.
  • • 
    The L. pimpinellifolium parent fruit had higher fruit invertase activities, greater hexose contents and lower sucrose accumulation that the L. esculentum parent. A strong correlation between fruit invertase activity and soluble sugar content was observed only in the L. pimpinellifolium parent and not in the homozygous fruit from either cross. In all cases high soluble acid invertase activities prevented sucrose accumulation and led to the build-up of hexoses.
  • • 
    The introgression of the invertase locus from L. pimpinellifolium into L. esculentum did not result in higher soluble solids in the progeny than in the L. esculentum parent, but it did modify their relative composition. Changes in the invertase gene composition alone are not sufficient to increase fruit soluble solids in tomato.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Current tomato breeding programmes target tomato fruit yield and quality. Carbohydrate content and composition are important factors determining tomato fruit quality and flavour (Stevens et al., 1977) and are major components of the soluble solids content. The latter is of greatest interest to the processing industry and is fundamentally determined by the fruit invertase content.

Invertases, or sucrose hydrolases, are considered to be present in most if not all plants (ap Rees, 1974; Avigad, 1982). Tomato (Lycopersicon species) has been divided into two subspecies; these are the green-fruited species (eulycopersicon) that accumulate sucrose, and the red-fruited species (eriopersicon) that accumulate hexoses in the form of glucose and fructose (Davies, 1966; Manning & Maw, 1975). Lycopersicon chmielewskii; Lycopersicon peruvianium and Lycopersicon hirsutum are examples of the former whilst Lycopersicon esculentum and Lycopersicon pimpinellifolium both fall into the latter category. L. peruvanianum (Stommel, 1992), L. chmielewskii (Yelle et al., 1988) and L. hirsutum (Miron & Schaffer, 1991) have lower soluble acid invertase contents compared with the hexose-accumulating L. esculentum. In green-fruited species, the onset of sucrose accumulation is characterized by low soluble acid invertase activity (Yelle et al., 1988; Miron & Schaffer, 1991). Walker et al. (1978) have suggested that sucrose hydrolysis via the acid invertase pathway may determine the extent and the rate by which sucrose (as opposed to glucose and fructose) is accumulated in tomato fruit.

More evidence for the involvement of soluble acid invertase in sucrose accumulation comes from studies with genetically altered tomatoes. Yelle et al. (1991) and Klann et al. (1993) used tomato fruit produced from crosses between L. esculentum and L. chmielewskii (a sucrose-accumulating fruit) to investigate the contribution of acid invertase activity to sucrose content. Klann et al. (1993) used both marker-assisted selection and back-crossing to show that a soluble acid invertase gene from L. chmielewskii mapped to the same locus as the sucrose-accumulating trait. The ability to store sucrose in the vacuole was found to be associated with low activities of soluble acid invertase (Yelle et al., 1991; Klann et al., 1993). More importantly, the sucrose-accumulating trait (or low acid invertase activity) was transferred genetically from one type of tomato to another. Transformed tomato fruit with an invertase cDNA in the antisense orientation or with a short invertase cDNA in the sense direction had markedly lower acid invertase activities than the controls (Ohyama et al., 1995; Klann et al., 1996). The decrease in acid invertase activity correlated with an increase in the relative amount of sucrose present. This provides very strong evidence that the presence of high soluble acid invertase activities prevents sucrose accumulation and leads to hexose accumulation in tomato fruit.

Recent genetic analysis in L. pimpinellifolium has shown that the locus conferring high soluble solids coincides with the region containing the known position of vacuolar invertase (Grandillo & Tanksley, 1996; Tanksley et al., 1996). Invertase mRNA from L. pimpinellifolium LA 722 fruit was present earlier in development than that from L. esculentum fruit (Elliot et al., 1993). This indicates that invertase may be important in the observed high sugar content of the former fruit. However, work in our laboratory has recently found that the same strain of L. pimpinellifolium LA 722 had neither a greater invertase activity nor a greater sugar content than L. esculentum (Husain, 1999).

The study described in this paper was undertaken to increase the soluble solid content of tomato fruit by genetic manipulation of invertase. An L. pimpinellifolium accession containing high invertase activity was crossed with L. esculentum, that has relatively low activity. The traits associated with the transfer of an invertase gene from the former parent into the latter are described with the aim of ascribing a role for invertase in tomato fruit sugar metabolism.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Growth of plant material

F1 progeny of a cross between L. esculentum FM 6203 and L. pimpinellifolium PI 126436 were backcrossed 5 times to L. esculentum and then selfed to produce the BC 5S1 generation. Individual plants were selected by RFLP analysis (data not shown) that were either homozygous for the invertase allele from L. esculentum (ee) or from L. pimpinellifolium (pp).

Seeds from L. esculentum, the nonrecurrent the parent L. pimpinellifolium and the ee and pp families were planted in humax compost in seed trays and grown in a growth room under a 12-h photoperiod at 20°C for approx. 4 wk. Plants were watered every 2 d and the compost kept moist. They were then transferred to pots in a glassnhouse under a 16-h photoperiod at 20–25°C and grown until the fruit were fully ripe and red. Flowers were tagged at the onset of anthesis and the plants were watered twice a day and fed from the appearance of the first truss with Tomorite® (Levington Horticulture Limited, Ipswich, UK) tomato feed every 2 d. Fruit was harvested every 7 d, weighed, frozen immediately in liquid nitrogen, ground to a powder and stored at −80°C for further analysis.

Sugar extraction and analysis

The sugars sucrose, glucose and fructose were extracted according to the method of Lyne & ap Rees (1971) with minor modifications. Aliquots of frozen tomato powder (20–25 mg) were boiled in 300 µl 80% (v/v) ethanol for 3 min and then centrifuged at 10 000 g for 15 min at 4°C. The resulting pellet was extracted a further three times in 80% (v/v) ethanol at 60°C for 10 min and the combined supernatants taken to dryness using a vacuum desiccator and freeze drier. The resulting residue was re-dissolved in deionized water.

Glucose was measured using the coupled assay system as previously described by Bergmeyer et al. (1974), with minor modifications. Aliquots of supernatant (10–20 µl) were added to reaction mixtures (200 µl) containing 100 mM Hepes (NaOH) pH 7.0, 10 mM MgCl2, 10 mM NAD+, 10 mM ATP and 1 U ml−1 glucose-6-phosphate dehydrogenase (Sigma), with 0.2 U hexokinase (Sigma) added to each sample to start the reaction. Fructose was assayed in the same extract by the addition of 0.2 U phosphoglucose isomerase (Sigma) while sucrose was determined in the same extract by the addition of 0.2 U invertase (BDH, Poole, Dorset, UK). The production of NADH in this reaction was followed at 340 nm using a microtiter plate reader at 37°C with intergral software until no further change was observed to determine the end point of the reaction. The sugar content was determined using glucose, fructose and sucrose standards with every set of samples.

Invertase extraction and assay

Invertase was extracted according to the method of Stommel (1992) with minor modifications. Aliquots of frozen tomato powder were extracted three times in 100 µl of extraction buffer containing 50 mM Hepes (NaOH) pH 7.5, 5 mM MgCl2, 1 M NaCl, 1 mM sodium EDTA, 0.5 mg ml−1 BSA, 5 mM DTT and 0.1% (v/v) Triton x-100. Extracts were microfuged at 10 000 g for 10 min at 4°C and the supernatants were then desalted and concentrated using microconcentrators (10 000 MW cut off; Amicon, Beverly, MA, USA). The concentrates were diluted with 50 mM Hepes (NaOH) pH 7.5, 5 mM MgCl2 and 5 mM DTT to 100 µl and used to determine the total soluble invertase activity. This included both vacuolar and apoplastic forms. The pellets were resuspended in 50 mM Hepes (NaOH) pH 7.5, 5 mM MgCl2 and 1 mM sodium EDTA and used to determine the ‘insoluble’ invertase activity.

Invertase activity was assayed in both the soluble and insoluble fractions by a stopped reaction procedure as described in Stommel (1992) with minor modifications. All steps were carried out on a microtiter plate. For soluble invertase, 3 µl extract was incubated with 15 µl of 0.1 M sodium citrate : 0.2 M Na2HPO4 buffer pH 4.0 and 12 µl 0.6 M sucrose for 30 min at 37°C in a total reaction volume of 30 µl. After incubation, 24 µl 1 M imidazole (HCl), pH 7.0 was added and the reaction stopped by boiling for 3 min. The rate of the reaction was determined from the amount of glucose and fructose produced in the 30-min incubation period as described above except that glucose plus fructose standards were used. Controls, using boiled enzyme extract were run for every sample and gave no absorbance (data not shown).

For insoluble invertase, the reaction mixture consisted of 12 µl extract, 15 µl citrate : phosphate buffer pH 4.4 and 3 µl 1.8 M sucrose in a total reaction volume of 30 µl. After 30-min incubation at 37°C, 24 µl 1 M imidazole (HCl), pH 7.0 was added and the reaction stopped by boiling for 3 min. The reaction mixture was then centrifuged at 10 000 g for 15 min. The rate of the reaction was determined as described for soluble invertase.

Statistical analysis

Statistical analysis was carried out using the linear and nonlinear regression and ANOVA tools of Microsoft Excel® (Microsoft Limited, Reading, UK) and Sigma Plot® (SPSS Inc., Woking, UK). For invertase-sugar relationships, a significance value of P < 0.01 gives a probability that a correlation coefficient between two variables arises by chance is < 1%.

Results and Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

Molecular approaches are being used increasingly to investigate the role of sugar-metabolizing enzymes in sink development and sugar signalling. Invertases have been shown to have a major function in early plant development and in sucrose partitioning in carrot (Sturm, 1999; Tang et al., 1999). In contrast, sucrose synthase activity was required for fruit set in tomato (D’Aoust et al., 1999). Sucrose synthase, rather than invertase, controls sucrose import into developing fruit for the first 10 d after anthesis (D’Aoust et al., 1999). Thereafter, invertase activity becomes increasingly important in fruit sugar metabolism but its precise functions remain unclear. Progeny of a cross between a low-invertase species (L. esculentum FM 6203) and a high fruit invertase species (L. pimpinellifolium PI 126436) were homozygous for either the L. pimpinellifolium invertase gene (pp) or the L. esculentum invertase gene (ee). Both showed the same fruit phenotype (Fig. 1) and also the same plant phenotype. In contrast, parent L. pimpinellifolium plants were much bushier and the tomato fruit much smaller, reaching only about 1 cm diameter when fully ripe (not shown).

image

Figure 1. Tomato fruit produced from the BC 5S1 generation of a cross between Lycoperisicon pimpinellifolium and Lycoperisicon esculentum. Fruit was produced that was either homozygous for the invertase allele from L. esculentum (ee; left hand side) or the invertase allele from L. pimpinellifolium (pp; right hand side).

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Fruit from L. pimpinellifolium PI 126436, ee, and pp all showed sigmoidal growth curves (Fig. 2). The maximum fresh weight of tomato fruit from both ee (Fig. 2c) and pp (Fig. 2b), however, were up to 40 times greater than that for ripe fruit from L. pimpinellifolium (Fig. 2a). These values were similar to those of ripe fruit from L. esculentum (38.5 ± 6.1; Husain, 1999). There was no significant difference between the f. wt of ripe ee fruit and ripe pp fruit (Table 1). The L. pimpinellifolium parent fruit had higher fruit invertase activities (Fig. 3), greater hexose contents (Fig. 4a) and lower sucrose accumulation (Fig. 4b) than the L. esculentum parent.

image

Figure 2. Changes in fruit weight during the development of Lycoperisicon pimpinellifolium PI 126436 (a) and the BC 5S2 families pp (b) and ee (c). The values are the mean of three measurements ± SE. Where no error bars are shown, the size of the error is less than that of the symbol.

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Table 1.  Sugar and invertase composition of red Lycoperisicon pimpinellifolium PI 126436, pp and ee fruit
 L. pimpinellifolium PI 126436ppee
  1. Glucose, fructose and sucrose contents, and soluble and insoluble acid invertase activities were determined in fruit during development. Values represent the mean of nine measurements ± SE. An asterisk indicates when P < 0.01.

Fresh weight (g)   1.2 ± 0.1 39.5 ± 0.7* 31.5 ± 5.1*
Sugar content (µmol g −1 f. wt)
Glucose 170.3 ± 14.3 57.3 ± 15.5* 66.4 ± 8.7*
Fructose 213.9 ± 17.1 76.2 ± 18.2* 81.0 ± 15.4*
Sucrose   7.1 ± 3.1  1.7 ± 0.2* 11.6 ± 1.1
Hexose 384.2 ± 3.1133.4 ± 32.9*147.3 ± 23.3*
Total Sugar 397.2 ± 31.0135.1 ± 32.8*158.0 ± 24.4*
Hexose : sucrose  60.9 ± 21.2 82.0 ± 26.3 12.7 ± 0.8*
Acid invertase activity (µmol hexoses produced g −1 f. wt h −1 )
Soluble1948.1 ± 127.7855.7 ± 234.6969.5 ± 222.6
Insoluble  64.4 ± 13.1 27.1 ± 5.8 37.7 ± 4.3
image

Figure 3. Activity of acid invertase in the parents Lycoperisicon esculentum (a) and Lycoperisicon pimpinellifolium (b) fruit. Soluble acid invertase activity was determined during fruit development. The values are the mean of three measurements ± SE. Where no error bars are shown, the size of the error is less than that of the symbol.

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image

Figure 4. Analysis sugar content of the parents Lycoperisicon esculentum (i, top) and Lycoperisicon pimpinellifolium (ii, bottom). Hexose (a) and sucrose (b) were determined during fruit development. The values are the mean of three measurements ± SE. Where no error bars are shown, the size of the error is less than that of the symbol.

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Sugar content and composition

Numerous measurements of the development-related changes in soluble sugars in different tomato species and cultivars are found in the literature (e.g. Stommel, 1992; Klann et al., 1993; Wang et al., 1993). These studies demonstrate that the evolution of fruit sugars depends more on genotypic differences between cultivars and species than changes in invertase-sugar relationship alone. In our own work, for example, clear differences in the invertase-sugar relationships between two cultivars of the same species (L. pimpinellifolium PI 126436 and LA 722) were found (Husain, 1999). These differences preclude effective comparison between results from different studies. For this reason, we have not attempted to extensively compare the results presented here and those previously described (Stommel, 1992; Klann et al., 1993; Wang et al., 1993).

The changes in fruit glucose, fructose and sucrose during development are shown in Fig. 5. The glucose content of fruit from the parent L. pimpinellifolium PI 126436 fruit significantly (P < 0.01) increased with fruit development (Fig. 5ai). This was also seen in ee fruit (Fig. 5ci), although the increase was much less than in the L. pimpinellifolium PI 126436 cultivar (compare gradients in Fig. 5ai & 5ci). There was no increase in glucose content in pp fruit during development (Fig. 5bi). Red ripe fruit from L. pimpinellifolium PI 126436 contained significantly (P < 0.01) more (over 250%) glucose than equivalent fruit from ee or pp plants (Table 1). Moreover, there was no difference between the mean glucose content of ee and pp fruit (Table 1).

image

Figure 5. Analysis of individual sugar contents of Lycoperisicon pimpinellifolium (a), pp (b) and ee (c) fruit. Glucose (i, top), fructose (ii, middle) and sucrose (iii, bottom) were determined during fruit development. The values are the mean of three measurements ± SE. Where no error bars are shown, the size of the error is less than that of the symbol.

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The fructose content of fruit produced from L. pimpinellifolium PI 126436 and ee plants also significantly (P < 0.01) increased with development (Fig. 5aii & 5cii), although again the increase in ee plants was relatively small. There was no significant increase in fructose content during the development of pp fruit (Fig. 5bii). Like the glucose content of red fruit, the mean fructose content of red L. pimpinellifolium PI 126436 was significantly (P < 0.01) greater (over 250%) than that found in red fruit from ee or pp fruit (Table 1). There was no difference in mean fructose content between red fruit from ee and pp (Table 1). In fruit from L. pimpinellifolium PI 126436, mean fructose content of red fruit was significantly (P < 0.01) greater (25%) than the mean glucose content (Table 1) of the same fruit. This was not the case in ee or pp fruit, where there was no significant difference (Table 1).

The sucrose content of L. pimpinellifolium PI 126436 fruit significantly (P < 0.01) increased with development (Fig. 5aiii). It decreased (P < 0.01) during the development of pp fruit (Fig. 5biii), while there was no change in the sucrose content during the development of ee fruit (Fig. 5ciii). The most significant difference between the ee amd pp lines appears to be that ee fruit have a much higher sucrose content than pp fruit (Fig. 6biii, 6ciii). The mean sucrose content of L. pimpinellifolium PI 126436 or ee red fruit (between which there was no difference) was significantly (P < 0.01) greater (400–600%) than that found in red fruit from pp plants (Table 1). In all cases, hexoses (glucose and fructose) were the dominant storage sugars (Table 1).

image

Figure 6. Relationships between soluble acid invertase activity and sugar content and composition in Lycoperisicon pimpinellifolium (a), pp (b) and ee (c) fruit. The relationships between soluble acid invertase activity and hexose (i, top), total sugar (ii, upper middle), sucrose (iii, lower middle) and hexose to sucrose ratio (iv, bottom) were determined in developing fruit. The values are the mean of three measurements ± SE. Where no error bars are shown, the size of the error is less than that of the symbol.

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Fig. 6 shows the changes in total sugar content and the hexose to sucrose ratio in the developing fruit. The total sugar content of L. pimpinellifolium PI 126436 and ee fruit also increased ( P  < 0.01) with fruit development ( Fig. 7ai, 7ci ). This was not observed in pp fruit ( Fig. 7bi ). Furthermore, the mean total sugar content of ripe L. pimpinellifolium PI 126436 fruit was greater (250–300%; P  < 0.01) than that found in red fruit from both ee or pp fruit ( Table 1 ). Again, there was no difference between the total sugar content of ee and pp red fruit ( Table 1 ).

image

Figure 7. Analysis of total sugar content and composition of Lycoperisicon pimpinellifolium (a), pp (b) and ee (c) fruit. Total sugar (i, right hand side) and the hexose to sucrose ratio (ii, left-hand side) were determined during fruit development. The values are the mean of three measurements ± SE. Where no error bars are shown, the size of the error is less than that of the symbol.

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Fruit produced from pp plants showed a positive correlation (P < 0.01) with the hexose to sucrose ratio and fruit development (Fig. 7bii). This was in contrast to L. pimpinellifolium PI 126436 and ee fruit, which both showed no significant correlation between the hexose to sucrose ratio and fruit development (Fig. 7aii, 7cii). Both L. pimpinellifolium PI 126436 and pp red fruit had a hexose : sucrose ratio value significantly (P < 0.01) greater (500–600%) than that found in ee fruit (Table 1). This reflects the much lower sucrose content of pp fruit rather than a difference in hexose content. There was no difference in the mean hexose : sucrose ratio values between L. pimpinellifolium PI 126436 and pp red fruit (Table 1).

Invertase activity

In the present study, total soluble invertase activity was assayed and no attempt was made to distinguish between apoplastic and vacuolar isoforms. Insoluble invertase activity followed similar trends to that of the soluble form (data not shown) and is therefore not discussed further. In spite of the fundamental difference in the invertase gene composition between the progeny, pp fruit did not have a greater soluble acid invertase activity than ee plants. The developmental profiles of soluble invertase activity found in both pp and ee fruit were similar to those found for L. esculentum FM 6203 (Fig. 3). A characteristic increase in activity was observed between 30 and 50 d after anthesis. In contrast, L. pimpinellifolium PI 126436 fruit showed a linear increase in soluble invertase activity during development (Fig. 3, Fig. 8). This was absent from fruit containing either invertase gene.

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Figure 8. Activity of acid invertase in Lycoperisicon pimpinellifolium (a), pp (b) and ee (c) fruit. Soluble acid invertase activity was determined during fruit development. The values are the mean of three measurements ± SE. Where no error bars are shown, the size of the error is less than that of the symbol.

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The changes in invertase activity during the development of the three types of tomato fruit are shown in Figs 3 and 8. The soluble acid invertase activity of L. pimpinellifolium PI 126436 fruit increased steadily (P < 0.01) during development (Figs 3, 8ai). This was not the case during the development of ee or pp fruit, where there was a sharp increase in activity between 30 and 40 d after anthesis (Fig. 8bi, 8ci). Interestingly, acid invertase activity increases earlier in the L. pimpinellifolium fruit than in either the pp or ee fruit. Invertase acitivity increased slightly earlier in pp fruit than in ee fruit (Fig. 8). There was a slight decline in activity during the final developmental stage (between 50 and 60 d after anthesis) in both cases. Mean soluble acid invertase from red L. pimpinellifolium PI 126436 was greater (over 200%; P < 0.01) than similar mean values for ee and pp fruit (Table 1). There was no difference between the mean soluble acid invertase activity of red fruit from ee plants and pp plants (Table 1).

Invertase is controlled by a complex array of transcriptional and post-translational controls. There are four specific cDNAS encoding extracellular invertases from tomato and these are characterized by highly differential sink tissue expression patterns (Godt & Roitsch, 1997). In addition there are at least two vacuolar invertase genes in tomato. The results in this manuscript demonstrate that transfer of a portion of chromosome 3 containing an invertase locus alone is insufficient to modify tomato fruit carbohydrate metabolism. Presumably, this is because of endogenous regulation that prevents the genetically programmed increases in invertase activity. Post-transcriptional control of invertase activity by interaction with an endogenous proteinaceous invertase inhibitor has been described in tomato and other species (Greiner et al., 2000). The activity of invertase is therefore regulated in a way that cannot be controlled simply by the movement of invertase genes.

Elliot et al. (1993 ) found only one invertase gene from L. esculentum and L. pimpinellifolium fruit on southern blots. Moreover, the abundance of invertase transcripts increased at an earlier stage of development in L. pimpinellifolium than L. esculentum , in agreement with the earlier increase in activity in the former than the latter observed here ( Fig. 3 , Elliot et al., 1993 ). We are currently testing the hypothesis that an endogenous regulatory inhibitor protein determines invertase activity in these tomato fruit.

Since the cross involved the transfer of a large segment of chromosome 3, it is possible that other factors influencing invertase activity were transferred with the structural gene for invertase. If the invertase locus introgressed into pp fruit is the gene responsible for a high soluble invertase activity in L. pimpinellifolium PI 126436, then other factors (such as activators and/or inhibitors) must influence for complete expression of the gene (in terms of in vivo activity).

The relationship between sink strength and invertase activity observed in L. pimpinellifolium is not transferrred to any progeny

The very strong correlation between invertase and soluble sugars in the L. pimpinellifolium parent indicates a cause-and-effect relationship (Fig. 6aii). This would suggest that soluble acid invertase activity plays a role in determining sink strength in L. pimpinellifolium PI 126436, but not in ee or pp fruit. No correlations were observed with hexose, total sugar content or sucrose content or hexose to sucrose ratio the L. esculentum parent (data not shown). Fruit hexose and total sugars were positively (P < 0.01) correlated with soluble acid invertase activity in these fruit throughout development only in the L. pimpinellifolium parent (Fig. 6ai, ii). Neither fruit sucrose nor the hexose to sucrose ratio were correlated with soluble invertase activity in any fruit (Fig. 6).

There was no correlation between soluble acid invertase activity and the hexose content (Fig. 6bi) or the total sugar content (Fig. 6bii) observed in pp fruit. It is important to note that the fruit sucrose content (Fig. 6biii) decreased (P < 0.01) with increasing invertase activity in the pp system. This is reflected in the hexose to sucrose ratio (Fig. 6biv) which increased (P < 0.01) with invertase increasing activity. The absence of a correlation between soluble acid invertase activity and either fruit hexose or total sugars strongly indicates that, in the pp plants, soluble acid invertase activity is less involved in sugar accumulation than in the L. pimpinellifolium PI 126436 or in ee fruit.

The parent cultivar, L. pimpinellifolium PI 126436 clearly shows a positive (P < 0.01) correlation with total sugar accumulation (Fig. 6aii). Therefore, the presence of an invertase gene from L. pimpinellifolium PI 126436 alone does not confer the trait leading to a positive relationship between higher soluble solids content and invertase activity. This suggests that multiple genes influence sugar accumulation in tomato fruit and that other factors in the cell are needed for a full expression of the invertase encoded by the introduced genes.

However, it appears that soluble acid invertase plays a role in sucrose accumulation in pp fruit. The negative correlation found between invertase activity and fruit sucrose strongly suggests that, in this fruit, sucrose only substantially accumulates in the absence of high soluble acid invertase activity. This characteristic has been observed previously in sucrose-accumulating tomato fruit (Yelle et al., 1988; Miron & Schaffer, 1991; Yelle et al., 1991; Klann et al., 1993).

Fruit from pp plants showed soluble invertase-sugar relationships different to those found in both L. esculentum FM 6203 and L. pimpinellifolium PI 126436. The introgression of the invertase locus from L. pimpinellifolium PI 126436 into L. esculentum FM 6203 did not result in higher soluble solids than the L. esculentum parent, but it did modify their relative composition as shown by the hexose : sucrose ratios. This clearly indicates that the strategy employed in this study was too simple and that changes expression of invertase alone is not sufficient to increase soluble solids, since the activity of the product of the transgene is controlled by many factors.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References

This paper is written in memory of Professor Tom ap Rees, who started this work. S. E. Husain is particularly grateful to Tom, who died tragically in the first year of her Ph.D. studies from which this manuscript is derived. The authors thank Elaine Higgins and Lou Dehn for the production of the BC5S1 seeds.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Acknowledgements
  7. References
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  • Davies JN. 1966. Occurrence of sucrose in the fruit of some species of Lycopersicon. Nature 209: 640641.
  • D’Aoust MA, Yelle S, Nguyen-Quoc B. 1999. Antisense inhibition of tomato fruit sucrose synthase decreases fruit setting and the sucrose unloading capacity of young fruit. Plant Cell 11: 24072418.
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