Ali, Hazem Abd El-Rahman Obiadalla: Understanding of Carbon Partitioning in Tomato Fruit

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Kapitel 5. Analysis of the Function of Chloroplastic Fructose 1,6-bisphosphatase in Tomato Fruit

5.1 Introduction

Fructose-1,6-bisphosphatase (FBPase) catalyses the inter-conversion of Fru-1,6-P2 and Fru-6-P. In green plant tissues there are two isoforms, one situated in the plastid (cp-FBPase), and one in the cytosol (cy-FBPase). The plastidial isoform is an important enzyme in control of the Calvin cycle, and its repression in potatoes leads to an inhibition of photosynthesis and a reduction in growth (Kossmann et al., 1994). The cy-FBPase isoform, on the other hand, is involved in gluconeogensis and sucrose synthesis. Inhibition of this isoform leads to increases in starch and decreases in sucrose synthesis (Zrenner et al., 1996; Strand et al., 2000).

Green tomato fruits contain photosynthetically active chloroplasts, which differentiate to chromoplasts during the ripening process. The cp-FBPase is present in green, but not in red fruits, which correlates with a switch from the fruits being photosynthetically active to becoming inactive (Büker et al., 1998). Fruits obtain sugars both directly from photosynthesis, and through import from source leaves via the phloem. The triose phosphate and glucose phosphate transporters are active in tomato chloroplasts (Büker et al., 1998), indicating that they could in principle both import and export sugars. It is not clear, therefore, what the role of fruit photosynthesis in fruit metabolism is, although it has been estimated to contribute between 10-15% of carbon skeletons in green fruits (Tanaka et al., 1974).

5.2 Aim of the work

The aim of this work is to study the role of cp-FBPase in tomato fruit metabolism. To accomplish this, tomato plants were transformed with a construct designed to repress cp-FBPase activity solely in the fruit. Transgenic lines were isolated with reduced amounts of cp-FBPase protein, which showed a reduction in total FBPase activity also. Fruits from these plants were analysed for alterations in carbohydrate metabolism.

5.3 Results

5.3.1 Recovery of Plants with Reduced FBPase Activity in the Pericarp of Tomato Fruit.

The pericarps from 25 days after flowering (DAF) old tomato fruits were analysed for FBPase activity in sixty transgenic lines. Three lines (#19, 33 & 34) were selected which showed


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reductions in FBPase activity (see Material and Methods). These cp-FBPase antisense plants were phenotypically identical to the untransformed control (Fig. 9).

Figure 9: Aerial parts of plants in both WT control and alpha-cp-FBP-transgenic lines after 8 weeks growth in the glasshouse. From left to right: untransformed WT control, alpha-cp-FBP#19, alpha-cp-FBP#33, alpha-cp-FBP#34 and alpha-cp-FBP#34. The alpha-cp-FBP plants are phenotypically identical to the untransformed WT control.

Fructose-1,6-bisphosphatase (FBPase) activity was studied throughout fruit development in the pericarp of these plants. In young (25 DAF), green, fruits, there was a significant reduction in total FBPase activity in all three transgenic lines in comparison with the WT control (Fig. 10A). At that time point the reduction in activity of line #34 was 25%, and of the other two lines 50%. The activity in all lines decreased during the ripening process, and was significantly lower than the WT control in lines #19 and #33 until 55 DAF when there was no significant difference.

Immunoblots using an antibody raised against wheat cp-FBPase (Hagelin et al., 1996), but which recognizes the tomato cp-FBPase also, indicated that it was completely eliminated in 25 DAF fruit of the lines #19 and #33, and greatly reduced in the line #34 (Fig. 10B). To demonstrate that the antisense effect was fruit specific, FBPase activity was also determined


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in the leaves of the transgenic plants. No differences between the transgenic plants and the WT control were found (Fig. 10C).

5.3.2 Starch and soluble sugar contents in the pericarp of the WT and transgenic lines

Starch and soluble sugar contents were determined in the pericarp between 25-70 DAF. There was net degradation of starch over the ripening period (Fig. 11A). In the control the concentration was approximately 8µmol hexose (g FW)-1 at 25 DAF, and was less that 1µmol hexose (g FW)-1 at the final sample point. The starch contents in lines #19 and #33 were not significantly different in comparison with the control over the entire sampling period, but those of line #34 were significantly (P= 0,05) reduced until 45 DAF, when there was no difference.

Glucose concentrations were significantly (P= 0,05) increased in the three transgenic lines in comparison with the WT control, but only between 25 and 35 DAF. At these time points the concentrations in the transgenic fruits were between 60-80 µmol hexose (g FW)-1, while in the WT control fruits they were between 40-50 µmol (g FW)-1 (Fig. 11B). After this point there were no significant differences with the concentration of glucose increasing to just less than 100µmol hexose (g FW)-1 at the final sampling point.

Fructose concentrations showed a similar pattern to glucose concentrations (Fig. 11C). They were initially about threefold greater in the transgenic lines being generally between 50-70 µmol hexose (g FW)-1 in comparison to under 20µmol hexose (g FW)-1 in the WT control fruits. The fructose concentration in the WT control fruits then increased to similar levels as in the transgenic plants by 45 DAF, and after this point the fructose concentration stayed relatively constant at between 60-80 µmol hexose (g FW)-1 in all the plants.

There were no significant differences in sucrose concentrations between the transgenic lines and WT control, except for two time points in one transgenic line. At 30 and 35 DAF in line #19 (Fig. 11D) there was greatly increased sucrose in comparison with the WT control. In all the other lines, however, the sucrose concentration decreased from about 3µmol hexose (g FW)-1 at the first sampling point, to about 1µmol hexose (g FW)-1 at the final sampling point.

5.3.3 Changes in activities in enzymes involved in conversion of sucrose to starch

Sucrose synthase (SuSy) activity decreased in pericarp tissue from WT control fruits over time from 176nmol min-1 (mg protein)-1 at 25 DAF to 62nmol min-1 (mg protein)-1 in the


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oldest fruit. In the transgenic lines the activity was significantly decreased at three time points (45, 55 and 65 DAF) in one transgenic line (#33; Fig 12A).

Figure 10: FBPase activity during developmental stage (A), Western blot analysis in green (25 DAF) (B) in the pericarp of WT and alpha-cp-FBP-transgenic lines [total soluble fruit protein (25µg) was subjected to SDS-PAGE on a 10% (w/v) gel] and FBPase activity in the leaves of WT control and alpha-cp-FBP-transgenic lines (C). Data represent the mean of five independent measurements + SE.


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Figure 11: Starch and soluble sugar contents in pericarp of WT and alpha-FBP-transgenic lines in tomato cultivar Moneymaker during development. (A) Starch. (B) Glucose. (C) Fructose. (D) Sucrose. Data represent the mean of five independent measurements + SE.


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UDP-glucose pyrophosphorylase (UGPase) activity was high in both the WT control and transgenic lines, and in the WT control the activity increased between 25 and 45 DAF, after which it decreased (Fig 12B). The activity in the fruits of the transgenic lines was significantly lower than the control at 25 DAF (line #33), 45 DAF (all lines) and 55 DAF (line #34; Fig 12B).

Phosphoglucomutase (PGM) activity decreased over the entire developmental period in the WT control from 1768nmol min-1 (mg protein)-1 at 25 DAF to 897nmol min-1 (mg protein)-1 at 65 DAF. The PGM activity was significantly reduced in the transgenic lines at 25 DAF (all lines), 35 DAF (line #19), 55 DAF (lines #19 and #34) and 65 DAF (lines #33 and #34; Fig 12C).

ADP-glucose pyrophosphorylase (AGPase) activity was significantly greater in the WT control at all time points up until 55 DAF than in all of the transgenic lines (Fig 12D).

5.3.4 Concentration of Metabolic Intermediates in the pericarp of the WT control and transgenic lines

The concentrations of several metabolites were measured in trichloroacetic acid extracts of the transgenic lines from 30DAF fruits. The metabolites determined were glucose 6-phosphate (Glc-6-P), glucose 1-phosphate (Glc-1-P), fructose 6-phosphate (Fru-6-P), 3-phosphoglyceric acid (3-PGA), phosphoenolpyruvate (PEP), pyruvate and inorganic phosphate (Pi). The data are presented in Table 1. The concentration of 3-PGA was increased in all lines, significantly (Ple0.05) so in lines #33 and #34. In addition the concentration of both Glc-1-P and Pi were significantly (Ple0.05) reduced in line #19. There were no other significant reductions of metabolite concentrations. The ratios of hexose phosphates (hexose-P) to 3-PGA, phosphate esters (P-ester) to Pi and 3-PGA to Pi were calculated also. The ratio of hexose-P to 3-PGA was reduced significantly (Ple0.05) in lines #33 and #19, but that of P-ester to Pi was increased, significantly in line #19. The ratio of 3-PGA to Pi was significantly (Ple0.05) increased in lines #19 and #34.

5.3.5 Analysis of fruit yield

Fruit were harvested, and their weights and sizes determined, after 65 DAF. Some fruits of the transgenic line can be seen in comparison with the WT control in Fig. 13. Both the average weights and sizes of fruits of all of the transgenic lines were significantly (P= 0.05) reduced in


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comparison with the WT control (Table 2). This reduction was between 15-20% with respect to the weights of the control fruits and 9-11% with respect to the sizes of the control fruits.

Figure 12: Activities of enzymes involved in the conversion of sucrose to starch in pericarp of the WT control and alphacp-FBP-transgenic lines of fruit of the tomato cultivar Moneymaker. (A) SuSy. (B) UGPase. (C) PGM. (D) AGPase. Data represent the mean of five independent measurements + SE.


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Table 1: Metabolite concentrations in the pericarp of 30 DAF old WT control and alphacp-FBP-transgenic fruits.

Conc. nmol.(g FW)-1

Lines

WT

#19

#33

#34

G6P

44.6 + 2.5

48.5 + 4.1

49.8 + 4.3

52.9 + 3.8

G1P

5.6 + 0.3

4.8 + 0.2

4.7 + 0.7

6.1 + 0.4

F6P

15.0 + 1.0

14.8 + 1.3

15.5 + 1.5

17.2 + 0.8

Total Hexose-P

65.2 + 3.1

68.1 + 4.4

70.0 + 5.8

76.2 + 6.3

3-PGA

15.0 + 1.0

18.4 + 1.0

19.3 + 2.2

19.7 + 1.5

PEP

4.5 + 0.6

3.5 + 0.4

4.6 + 0.4

5.1 + 0.8

Pyruvate

2.8 + 0.4

3.9 + 0.5

3.9 + 0.5

3.2 + 0.5

Pi

1.7 + 0.1

1.2 + 0.1

1.5 + 0.2

1.6 + 0.1

Ratio

 

 

 

 

Hexose-P/3-PGA

4.5 + 0.5

3.7 + 0.1

3.6 + 0.1

3.9 + 0.2

P-ester/Pi

50.6 + 5.3

79.4 + 12.6

59.8 + 6.8

60.2 + 2.6

3-PGA/Pi

8.8 + 1.1

15.3 + 3.4

12.8 + 3.9

12.3 + 0.9

The data represent means ± SE of five independent samples. Samples significantly different from the control (Ple0.05, Students t-test) are in bold.

Table 2: Weights and sizes of ripe tomato fruits in the WT control and alphacp-FBP-transgenic lines.

Lines

Average

Weight of Fruit (g)

Size of Fruit (cm)

WT

#19

#33

#34

54.2 + 1.2 (n=146)

46.4 + 2.1 (n=42)

45.8 + 1.8 (n=52)

43.5 + 1.7 (n=62)

4.8 + 0.03 (n=146)

4.4 + 0.08 (n=42)

4.4 + 0.07 (n=52)

4.3 + 0.06 (n=62)

The data are means + SE, number of sample is in parentheses. Significant differences (P= 0,05), Student‘s t-test are in bold


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Figure 13: Some 65 DAF old fruits from alphacp-FBP-transgenic lines (bottom) in comparison with a control fruit (above).(A) Transgenic line #19. (B) Transgenic line #33 (C) Transgenic line #34.


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5.3.6 Number of flower, fruit per plant, fruit set and number of days to 50% flowering.

Data of this trait are presented in Table 3. No significant differences were found between the control and the transgenic lines with respect to both number of flower per plant and number of days from planting to 50% flowering, while two transgenic lines (#19 and #33) showed significant reductions in comparison with the control with respect both number of fruit per plant and fruit set. No significant differences were found between the control and line #34 with respect to either number of flower per plant or fruit set.

Table 3: Number of flowers, fruits, fruit set and number of days to 50% flowering in the WT control and alphacp-FBP-transgenic lines.

Lines

Average

No. of flower/plant

No. of fruit/plant

Fruit set %

No. of days to 50% flowering

WT

#19

#33

#34

42.9 + 1.7

46.0 + 4.9

42.8 + 2.0

44.4 + 4.7

26.3 + 1.2

21.4 + 1.8

21.2 + 1.7

26.8 + 2.6

61.0 + 0.5

47.1 + 1.6

49.2 + 2.2

60.6 + 1.1

51.4 + 0.5

50.2 + 0.4

50.0 + 0.6

49.8 + 0.7

Data represent the mean of fifteen independent measurements + SE in WT and five independent measurements + SE in the transgenic lines. Significant differences (P= 0,05, Student‘s t-test) are in bold.

5.4 Discussion and conclusion

In this study I have described the production of transgenic tomato plants repressed in cp-FBPase activity in the fruit. A fruit specific promoter was used as constitutive reduction of this enzyme represses photosynthesis in leaves and lead to stunted growth of the whole plant (Kossmann et al., 1994). If plants had been produced in this study using a constitutive promoter it would not have been possible to separate the effect in fruit metabolism of reduced photosynthesis in the leaves from that in fruits. The transgenic plants appeared phenotypically normal, indicating that the antisense effect was indeed restricted to the fruits, a view confirmed by measurement of FBPase activity, which was unchanged in leaves of the transgenic lines. Although unchanged in the leaves, the total FBPase activity was reduced in green fruits. This is a measure of both cp-FBPase and cy-FBPase simultaneously. The reduction in comparison with the total activity was more than 50% in the most inhibited lines


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(#19 and #33), which is a smaller reduction in activity than was found in leaves of potato plants where the cp-FBPase was inhibited (up to 85% inhibition; Kossmann et al., 1994). This might indicate either that the cy-FBPase contributes a greater proportion of the total FBPase activity in tomato fruits than in potato leaves, or that the fruit specific promoter used in this study does not inhibit the cp-FBPase as strongly as the constitutive promoter used in the experiments of Kossmann et al. (1994). I feel that the former explanation is more likely as immunoblot experiments using an antibody that recognizes cp-FBPase indicated that it was almost completely eliminated in lines #19 and #33, and greatly reduced in lines #34. This indicates that the residual FBPase activity, at least in lines #19 and #33, comes almost entirely from the cy-FBPase. As expected the reduction in FBPase activity was found only in younger fruits (45 DAF or younger). It is known that the cp-FBPase is present in green, but not red, fruits and, therefore, that in red fruits all FBPase activity comes solely from the cy-FBPase (Büker et al., 1998). As it was the cp-FBPase isoform that was being repressed, differences in activity should have been noted only in younger fruits.

As was stated above, cp-FBPase activity has previously been repressed in transgenic potato plants and much data has been collected on alterations in metabolism in leaves of those plants. Repression of cp-FBPase in leaves led to decreases in the concentrations of soluble sugars and in starch contents (Kossmann et al., 1994). Such drastic differences were not found in the fruits of the transgenic tomato plants repressed in cp-FBPase and the differences that were noted were qualitatively different to those found in potato leaves. Glucose and fructose concentrations, for example, were not decreased in comparison with the control, but rather increased in green fruits. These alterations are at precisely the developmental stage when cp-FBPase would be expected to have the greatest influence indicating that it is indeed a reduction in activity of this enzyme that leads to the increase. This probably indicates that the fruits from the transgenic plants are relying more on imported sucrose than the WT control fruits. Sucrose is degraded very quickly upon import into fruits by either invertase or sucrose synthase. This is demonstrated by the very low concentration of sucrose in relation to either glucose or fructose found in the fruits both in the present study and in previous ones (Klann et al., 1996; Schaffer and Petreikov, 1997). Although it is not clear in tomato fruits which enzyme has the greatest influence on sucrose degradation, both appear to effect fruit metabolism. Repression of invertase leads to fruits that accumulate sucrose (Klann et al., 1996), whilst inhibition of sucrose synthase has been reported to decrease fruit set and sucrose import, but did not alter soluble sugar levels (D‘Aoust et al., 1999). Invertase degrades


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sucrose to glucose and fructose, whilst sucrose synthase produces UDP-glucose and fructose. Increases in concentrations of glucose and fructose in fruits of the transgenic lines indicates, therefore, either that more sucrose has been imported and degraded, or that the glucose and fructose produced are not being utilized as quickly. As the transgenic fruits are repressed in cp-FBPase, and it is known that repression of this enzyme leads to repression of photosynthesis (Kossmann et al., 1994), it is likely that the transgenic fruits will have to rely more on imported sucrose for growth than the WT control fruits. The levels of PEP and pyruvate were not altered in the transgenic lines, which indicates that glycolysis has not been down-regulated and, therefore, that utilization of glucose and fructose has also not been reduced. This indicates, therefore, that the increased concentrations of fructose and glucose are due to increased import of sucrose.

In one transgenic line (#19) at both 30 and 35 DAF there was a significantly greatly increased sucrose concentration in comparison with the WT control, and the other transgenic lines. I have no explanation for this, and it was not noted in any other line at any other point, indicating that it was due to random variation. Other than those points, there were no significant differences in sucrose concentrations in the transgenic lines in comparison with the WT control fruits. These lack of differences in soluble sugar concentrations between the transgenics and the WT control are presumably because the fruit can compensate for any reduction in sugar production due to a fruit specific repression in photosynthesis by importing more soluble sugars.

I also measured the concentrations of some metabolites, which might be affected by reductions in cy-FBPase (Table 1). To our knowledge these data represent the first documented measurement of these metabolites in tomato fruit. The levels of most measured metabolites are lower than those observed in leaves or tubers from the closely related potato plant (see for example Westram et al., 2002; Lytovchenko et al., 2002) most probably because of the high water content of this tissue. In addition, the relative 3-PGA concentration with respect to the other metabolites is much lower in the WT fruit tissue than that observed in potato leaves (Lytovchenko et al., 2002) suggesting that photosynthesis in the tissue studied here is less efficient. Although the levels of hexose-P were unchanged, the hexose-P to 3-PGA ratio - which is indicative of the rate of inter-conversion between Fru-1,6-P2 and Fru-6-P (Fernie et al., 2001) - is moderately decreased in all lines (significantly in the case of #33 and #19). This indicates that the flux through the chloroplastic FBPase is indeed inhibited in vivo. Furthermore, evaluation of the metabolic profile of these plants strongly hints that


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photosynthesis was repressed by phosphate limitation - the levels of inorganic phosphate (Pi) are somewhat lower in the transgenics (significantly in the strongest line), and most importantly the P-ester to Pi ratio increases. Such changes are characteristic of phosphate limitation of photosyntheis (Lytovchenko et al., 2002; Leegood and Furbank, 1986) and indeed resemble those observed on the inhibition of the cytosolic isoform of this enzyme in potato (Zrenner et al., 1996). The glycolytic metabolites, PEP and pyruvate were unaltered in the transgenics whilst there was a slight decrease in the levels of Pi. This was only significant, however, in the strongest transgenic line. The ratio of total P-ester to Pi shows a trend of increasing with decreasing cp-FBPase activity, as does the ratio of 3-PGA to Pi.

None of the metabolites downstream of the FBPase reaction (Glc-6-P, Fru-6-P and Glc-1-P) were greatly altered in the fruits of the transgenic plants. Glc-1-P was reduced, but only in the fruits from one line (#19). These data are similar to those reported by Kossmann et al. (1994) who argued that hexose-P are present mainly in the cytosol and, therefore, are less likely to be influenced by alterations in plastid metabolism. 3-PGA concentrations, however, were significantly increased in two out of the three transgenic lines. This is in contrast to what was found in potato leaves repressed in cp-FBPase where 3-PGA was reduced in concentration (Kossmann et al., 1994). In that study they did find, however, that glyceraldehyde 3-phosphate (G3P), the precursor of which is 3-PGA, was increased in leaves with reduced cp-FBPase. It was argued that photosynthetically active tissues with reduced cp-FBPase should contain less ribulose 1,5-bisphosphate (Ru 1,5-P2) which is the substrate for carboxylation. They argued further that it would be reasonable to assume that under those circumstances G3P would accumulate in preference to 3-PGA as G3P is involved in the regeneration phase of the Calvin cycle while 3-PGA is the primary product of carboxylation. Although these arguments hold true for leaves, where photosynthesis is essential for growth, they do not necessarily hold true for fruits, which have a second source of energy. As the fruit must not rely on its own photosynthate the regeneration phase of the Calvin cycle is not of such importance and, therefore, it is reasonable to assume that reductions in cp-FBPase would result in increases in 3-PGA.

The starch content in two out of three of the transgenic lines was not consistently altered in comparison with the WT control. One line (#34) did produce less starch than the WT control, but this was the line which was least inhibited in FBPase activity indicating that the reduction is not related to the reduction in cp-FBPase activity. Reduction in cp-FBPase activity in potatoes did lead to a reduction in starch accumulation in leaves (Kossmann et al., 1994).


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This differences seen in the fruits of the transgenic lines here in comparison with the situation in potato leaves probably due to a major difference between starch production in leaves and fruits. Tomato fruit chloroplasts contain both an active glucose phosphate and a triose phosphate transporter (Büker et al., 1998). Chloroplasts in leaves are not thought to contain a glucose phosphate transporter as, leaves do not appear to accumulate transcript that codes for this transporter (Kammerer et al., 1998). Repression of cp-FBPase activity should reduce flux of carbon produced by photosynthesis into starch. As no consistent reductions in starch were found in the transgenic lines it appears that fruit plastids can compensate for any reduction in flux from photosynthesis, presumably by importing glucose phosphate formed by cytoplasmic sucrose degradation. It appears that AGPase activity was reduced in the transgenic lines by up to 60 %. This is something that would be expected to reduce starch contents, but in the next chapter I will demonstrate that AGPase has to be repressed to a greater extent than was found in the transgenic lines before starch accumulation is affected. In addition the ratio of 3PGA to Pi in the pericarp of the transformants is elevated in the transgenic tomato lines (Table 1), which would be expected to stimulate AGPase activity and might compensate for any reductions in activity. In addition PGM activity was reduced in the transgenic lines at early stages of development. PGM exists as two isoforms, one in the cytoplasm and one in the plastid each of which contributes approximately 50 % of the total activity. It is known that the plastidial isoform is essential for starch biosynthesis (Hanson et al, 1988; Harrison et al., 1998; Tauberger et al., 2000), but it is not clear from my measurement which isoform is reduced in activity.

No significant differences were found between the WT control and all of transgenic lines with respect to either number of flower per plant or number of days from planting to 50% flowering. Two transgenic lines (#19 and #33), however, showed significant reductions in comparison with the WT control in respect to both number of fruit per plant and fruit set. These are the two most strongly inhibited lines indicating that this reduction in fruit set is indeed due to the reduction in FBPase activity. It is difficult to understand, however, how cp-FBPase could influence fruit set. It might be possible that its repression leads to reduced pollen viability or to alteration in ovule development. To my knowledge it is not known whether the B33 promoter confers expression in these tissues. If it did then this could be a feasible explanation. It has been demonstrated previously, for example, that reductions in citrate synthase activity in potato leads to disintegration of the ovary (Landschütze et al., 1995), indicating that alterations in metabolism can indeed influence fertility. A second


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possibility is that very young fruit abort for some unknown reason due to repression of cp-FBPase affecting carbohydrate metabolism within the fruit. These possibilities are something that could be investigated in the future.

One final question that I wished to address was how much carbon was supplied to the fruit through photosynthesis in the fruit itself. I have demonstrated above that cp-FBPase is repressed in the fruits of the transgenic plants, and it is known that this leads to reductions in photosynthesis (Kossmann et al., 1994). In addition, the metabolic analysis that I have carried out (discussed above) indicates that photosynthesis is indeed repressed in the fruits of these transgenic plants by phosphate limitation. The most likely explanation, therefore, for any effect on yield would be that it is caused by repression of photosynthesis in the fruits. Repression of cp-FBPase led to a reduction in both the weights and sizes of ripe fruits in all three transgenic lines (Fig. 13, Table 2). There are three possibilities as to how this could occur. The first is that the reduction in cp-FBPase leads to a reduction in sink strength in the fruits and, thus, to decreased import of carbon from the leaves. This seems unlikely, as it would be expected that reducing the amount of carbon produced by photosynthesis in the fruit would actually increase demand for carbon from the fruit. The second possibility is that in the transgenic plants there is less carbon fixed in the leaves and, thus, less exported to the fruits. This again appears unlikely as there was no effect of the transformation on FBPase activity in the leaves, indicating that the repression was fruit specific and, thus, that the only direct effects would be found in the fruits. The third, and most likely explanation is that the reduction in fruit weight represents the carbon produced within the fruit by photosynthesis. This explanation is supported as the reduction in average fruit weight was between 15-20% in the three lines. This is very similar to the estimate of the contribution of photosynthesis to production of carbon skeletons in green tomato fruits of Tanaka et al. (1974).

From the data presented in this chapter, it can be concluded that: (A) cp-FBPase activity is almost completely eliminated in the two most strongly inhibited lines (#19 and #33). (B) Repression of cp-FBPase in fruits leads to some alterations in concentrations of soluble sugars in young fruits. (C) The metabolic profile in fruits of the transgenic lines strongly indicates that photosynthesis is inhibited in vivo. (D) Fruit weight and size is reduced in the transgenic lines suggesting a significant contribution of fruit photosynthesis in the provision of carbon and energy required to support fruit expansion. (G) Repression of cp-FBPase affected fruit set, but the reason for this remains unclear.


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