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

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Kapitel 7. Analysis of the Function of the GWD protein in Tomato Fruit

7.1 Introduction

Starch is one of the most abundant polymers produced in nature and is synthesized as a storage carbohydrate throughout the plant kingdom. In storage organs it serves as a long-term carbon reserve, whereas in photosynthetically competent tissues it is transiently accumulated to provide both reduced carbon and energy during periods unfavourable for photosynthesis. Starch comprises both linear (amylose) and branched (amylopectin) glucose polymers. Amylopectin from many, but not all plant sources contains phosphate-monoesters that are linked mainly to the C6 and C3 positions of glycosyl residues. The biochemical mechanism of starch phosphorylation has, however, only recently been elucidated. Transgenic potato plants (Lorberth et al., 1998) and the sex1 mutant of Arabidobsis (Yu et al., 2001) are deficient in a starch associated protein, which was provisionally designated as R1, and they synthesise starch with a decreased phosphate content. The purified recombinant R1-protein from potato is able to phosphorylate alpha-glucans (Ritte et al., 2002). It catalyses a dikinase-type of reaction, liberating the gamma-phosphate of ATP (resulting in the release of orthophosphate), but using the beta-phosphate to phosphorylate glucosyl residues the polyglucan. Because of this the protein has been renamed as GWD (Glucan Water Dikinase; Ritte et al., 2003).

The phosphorylation of starch strongly affects its in vivo degradability. This is indicated by the starch-excess phenotype observed in leaves of GWD deficient potato or Arabidopsis plants (Lorberth et al., 1998; Yu et al., 2001). The reasons for this impairment of starch-degradation are, as yet, unknown.

7.2 Aim of the work

The role of starch in tomato fruits is not well understood. It accumulates in young fruits, but afterwards there is net starch degradation leading to it being almost undetectable in ripe fruits. One way of examining this is to alter how much starch accumulates during fruit development. In a previous chapter I described the repression of ADP-glucose pyrophosphorylase (AGPase) in tomato, which leads to reductions in starch accumulation. It would also be interesting, however, to take the reverse approach and study tomato fruits that accumulate starch over a longer time period than normal. As repression of the GWD protein leads to reductions in starch degradation I decided to try and accomplish this by manipulating the amount of GWD protein using genetic engineering techniques


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7.3 Results

7.3.1 Recovery of Tomato Plants with Repression of the GWD Protein

As was described in the Material and Methods, immunoblots demonstrated that three transgenic lines (#16, #17 and #20) showed a reduction in GWD protein accumulation (Fig. 20A) and were chosen for further study. The plants in all of these transgenic lines differed phenotypically form the untransformed control (Fig. 19). The leaves senesced much earlier than the control.

Figure 19: Aerial parts of plants in both WT control and alpha-GWD-transgenic lines after 8 weeks growth in the glasshouse. From left to right: WT control, transgenic line #16, transgenic line #17, transgenic line #20 and transgenic line #20.

To examine at what developmental period the GWD protein is present in tomato fruits, immunoblots was performed using protein extracts from differently aged WT fruits (Fig. 20C) and an antibody raised against the potato GWD protein (Ritte et al. 2000). This showed that it was present at every time point up to 50 DAF, after which it was absent. Immunoblots were also used to demonstrate that the protein was repressed in the pericarp of the transgenic plants


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also and it appeared to be completely absent from 25 DAF fruits in all of the transgenic lines (Fig 20B).

Figure 20: Immunoblot analysis of the GWD protein in (A) leaves of untransformed WT control and three selected transgenic lines [Total soluble leaf protein (15µg) was subjected to SDS-PAGE on an 8% (w/v) gel], (B) in the pericarp of the WT control and transgenic lines (25 DAF) [Total soluble fruit (pericarp) protein (30µg) was subjected to SDS-PAGE on an 8% (w/v) gel] and (C) in the pericarp of the WT control fruits between 25-70 DAF [Total soluble fruit protein (20µg) was subjected to SDS-PAGE on an 8% (w/v) gel].

7.3.2 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. 21A). In the control the concentration was approximately 8µmol hexose (g FW)-1 at 25 DAF, and was less that 0.5µmol hexose (g FW)-1 at the final sample point. The starch contents in all of transgenic


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lines were significantly reduced in comparison with the control until 45 DAF after which there were no differences.

There were no significant differences in glucose concentrations between the transgenic lines and control between 25 and 35 DAF. After that the concentrations were significantly decreased in the three transgenic lines in comparison with the WT. At these time points the concentrations in the transgenic fruits were between 40-50 µmol hexose (g FW)-1, while in the control fruits they were between 60-90 µmol (g FW)-1 (Fig. 21B).

Fructose concentrations were significantly increased in the three transgenic lines in comparison with the control, but only between 25 and 35 DAF. At these time points the concentrations in the transgenic fruits were between 40-55 µmol hexose (g FW)-1, while in the control fruits they were between 15-18 µmol (g FW)-1 (Fig. 21C). Between 40-70 DAF, however, the fructose concentrations in the transgenic lines were significantly decreased in comparison with the control. During this period the concentrations in the transgenic fruits ranged between 40-50 µmol hexose (g FW)-1, while in the control fruits they were between 60-100 µmol (g FW)-1.

Sucrose concentrations in the pericarps of all the transgenic lines were significantly lower than the control at virtually every time point. The exceptions were in line #20 at 30 DAF and line #17 at 65 and 70 DAF where the sucrose concentration was not significantly altered in comparison with the control (Fig. 21D) In the controls the sucrose concentration decreased from about 3µmol hexose (g FW)-1 at the first sampling point, to about 1.5µmol hexose (g FW)-1 at the final sampling point. Although variable, in the transgenic lines there was a general decrease in sucrose concentrations from approximately 1.5µmol hexose (g FW)-1 at 25 DAF to barely detectable levels at the final sample point.

7.3.3 Starch and soluble sugar contents in the leaves of the WT and transgenic lines

The starch and soluble sugar contents were determined also in the leaves of the control and transgenic plants (Fig.22A and B). The all of transgenic lines contained significantly more starch than the control (Sampling was done on fully developed leaves of 8 weeks old plants in the middle of the light period) (Fig.22A). In the control the concentration was 15.1µmol hexose (g FW)-1, but in the transgenic lines the concentrations were 26.5, 36,2, and 28,4µmol hexose (g FW)-1 for lines #16, #17 and #20 respectively.

There were significantly increased glucose concentrations in leaves of the transgenic lines in comparison with the control (Fig.22B). In the control the glucose concentration was 16.5µmol


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hexose (g FW)-1, but in all the transgenic lines it was approximately 21.0 µmol hexose (g FW)-1. Fructose concentrations conversely were significantly decreased in the three transgenic lines in comparison with the control (Fig.22B). The fructose concentration in the control was 22.2µmol hexose (g FW)-1, but in the transgenic lines they were 6.3, 6.6 and 7.0µmol hexose (g FW)-1 in lines #16, #17, and #20 respectively. Sucrose concentrations were significantly increased in the three transgenic lines in comparison with WT control (Fig.22B). In the control this was 3.6µmol hexose (g FW)-1, however, in the transgenic lines the concentrations were 6.5, 5.3 and 6.9µmol hexose (g FW)-1 in lines #16, #17, and #20 respectively

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

Sucrose synthase (SuSy) activity decreased over time in fruits of both the control and transgenic plants. Significant reductions in activities were found in the transgenic lines at 55 DAF (line #17) and 65 DAF (lines #16 and #20; Fig 23A). UDP-glucose pyrophosphorylase (UGPase) activity was high in both the control and transgenic lines, increasing between 25-55 DAF. It then decreased in activity between 55-65 DAF. The pericarp from transgenic lines #17 and #20 contained significantly reduced UGPase activity at 45 DAF else there were no significant differences between the control and transgenic lines (Fig 23B). Phosphoglucomutase (PGM) activity decreased over the developmental period in all the lines. There were significant reductions in activity of PGM in comparison with the control in all the transgenic lines at 25 DAF, in line #17 at 35 and 55 DAF, and in lines #17 and #20 at 65 DAF (Fig. 23C). ADP-glucose pyrophosphorylase (AGPase) activity decreased over time in the control from 39.3 nmol min-1 (mg protein)-1 at 25 DAF to 19.5 nmol min-1 (mg protein)-1 at 65 DAF (Fig. 23D). In the pericarp of the transgenic lines there was a significant reduction in AGPase activity at 25 DAF (lines #17 and #20), 35 DAF (line #20), 45 DAF (lines #17 and #20), 55 DAF (line #17) and 65 DAF (line #20). Fructose-1,6-bisphosphatase (FBPase) activity was generally greater in the WT control in comparison with the Ü-GWD-transgenic lines. The activity decreased slowly over time period in both WT control and Ü-GWD-transgenic lines (Fig. 23E).


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Figure 21: Starch and soluble sugar contents in the pericarp of the WT control and alpha-GWD-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 in the WT control and transgenic line #17, but four independent measurements + SE in transgenic lines #16 and #20


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Figure 22: Starch and soluble sugar contents in the leaves of the WT control and transgenic tomato lines lacking the GWD protein. (A) Starch. (B) Soluble sugars. Data represent the mean of five independent measurements + SE in the WT control and transgenic line #17, but four independent measurements + SE in transgenic lines #16 and #20.

7.3.5 Analysis of fruit yield

Fruits 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. 24. As can be seen in Table 7 both the average weights and sizes of fruits in all of the transgenic lines were significantly (P= 0.01) reduced in comparison with the control. This reduction was between 24-33% with respect the weights of the control fruits and 12-15% with respect the sizes of the control fruits.


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Figure 23: Activities of enzymes involved in the conversion of sucrose to starch in the pericarp of the WT control and alpha-GWD-transgenic lines of tomato cultivar Moneymaker.(A) SuSy. (B) UGPase. (C) PGM. (D) AGPase. (E) FBPase. Data represent the mean of five independent measurements + SE in WT control and transgenic line #17, but four independent measurements + SE in transgenic lines #16 and #20.


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Table 7: Weights and sizes of ripe tomato fruits in WT control and alpha-GWD-transgenic lines.

Lines

Average

Weight of Fruit (g)

Size of Fruit (cm)

WT

54.2 + 1.2 (n=146)

4.8 + 0.03 (n=146)

#16

36.1 + 1.8 (n=21)

4.1 + 0.08 (n=21)

#17

36.2 + 1.4 (n=31)

4.1 + 0.05 (n=31)

#20

40.9 + 1.3 (n=27)

4.2 + 0.05 (n=27)

Data are means + standard error, number of samples is in parentheses. Significant differences (P= 0,01), Students t-test are in bold.

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

Data of this trait are presented in Table 8. As can be seen in Table 8, the both average number of fruit per plant, fruit set and number of days to 50% flowering of all of the transgenic lines were significantly (P= 0.01) reduced in comparison with the WT control. This reduction was between 42-46% with respect the number of fruit per plant of the control fruits and 49-53% with respect the fruit set of the control fruits. Transgenic lines (#17) was significantly (P= 0.05) increased with respect number of flower per plant in comparison with the WT control, while no different significant were found between WT control and transgenic lines (#16 and #17) with respect number of flower per plant.

Table. 8: Number of flowers, fruit set and number of days to 50% flowering in the WT control and alpha-GWD-transgenic lines.

Lines

Average

No. of flower/plant

No. of fruit/plant

Fruit set %

No. of days to 50% flowering

WT

#16

#17

#20

42.9 + 1.7

47.5 + 3.3

53.2 + 3.2

49.5 + 4.4

26.3 + 1.2

14.8 + 2.3

15.2 + 1.4

14.0 + 1.6

61.0 + 0.5

30.8 + 3.3

28.6 + 1.8

28.1 + 1.0

51.4 + 0.5

54.8 + 1.3

56.2 + 0.9

57.0 + 1.4

Data represent the mean of fifteen independent measurements + SE in the control, five independent measurements + SE in line #17 and four independent measurements + SE in lines #16 and #20. Significant differences (P= 0.05 and P= 0.01, Student‘s t-test) are in bold.


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Figure 24: Some 65 DAF old fruits from alpha-GWD-transgenic lines (bottom) in comparison with the WT control fruit (above). (A) Transgenic line #16. (B) Transgenic line #17. (C) Transgenic line #20.


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7.4 Discussion and conclusions

In this study I have described the production of transgenic tomato plants repressed in the amount of GWD protein using a constitutive promoter. The GWD protein was demonstrated to be present in the pericarp of WT fruits only between 25-50 DAF, which is also the period when starch is present in the pericarp. This is consistent with the known role of the protein as an amylopectin phosphorylating enzyme. The protein was completely lacking in both leaves and pericarp of three transgenic lines chosen for further study.

Potato and Arabidopsis plants lacking the GWD protein do not degrade starch in their leaves as quickly as WT plants, leading to a starch-excess phenotype (Lorberth et al., 1998, Yu et al., 2001). This was also found in leaves of the tomato plants lacking GWD protein which contained approximately twice the starch content of the control. Interestingly, however, the starch content in the pericarp was decreased in the transgenic lines rather than increased. The most likely explanation for this comes from studying the phenotype of the transgenic plants. The leaves of these plants senesced extremely early in comparison with the WT control. This is presumably some form of stress response caused by the accumulation of large amounts of starch in the leaves. This response was not noted in potato plants lacking the GWD protein (Lorberth et al., 1998), indicating that tomato responds differently to potato when it is repressed in starch degradation. Arabidopsis plants lacking GWD protein do, however, grow significantly worse than controls under certain environmental conditions (Caspar et al., 1991; Trethewey and ap Rees, 1994b). In any case, the senesced leaves should also be inhibited in photosynthesis meaning that they would not be able to produce as much sugar to export to the fruits as the control. I demonstrated in a previous chapter that fruit specific repression of chloroplastic fructose-1,6-bisphosphatase (cp-FBPase) - which leads to fruit specific inhibition of photosynthesis - did not produce significant reductions in starch contents, indicating that fruit starch is mainly the product of sucrose imported from the leaves. As the fruits from the transgenic plants in this study should be receiving less sucrose than the control it is reasonable to assume that this is what causes the reductions in starch contents. In addition I found that the activity of ADP-glucose pyrophosphorylase (AGPase) was reduced in the transgenic fruits. AGPase has often been considered to be an important enzyme in determining starch contents in storage organs, including tomato fruits (Yelle et al., 1988), and decreases in its activity would be expected to lead to reductions in starch contents. The reasons for its reduction in activity are not clear, but it is reasonable to assume that it may have something to do with alterations in soluble sugar concentrations within the pericarp.


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Soluble sugar levels have often been considered to be important in regulating transcription of genes, and it has been demonstrated, for example, that AGPase transcript is greatly affected by growing Arabidopsis leaf discs on different soluble sugars (Sokolov et al., 1998). In the pericarp of the transgenic plants it was found that the concentrations of sucrose, fructose and glucose were lower than in the control at virtually every time-point measured indicating that starch synthesis may indeed be regulated in this way in tomato fruits.

In this study I also analysed the yield of tomato fruits from the lines and found the both average weights and sizes of fruits of all of the transgenic lines were significantly (P= 0.01) reduced in comparison with the WT control. This reduction was between 24-33% with respect the weights of the WT control fruits and 12-15% with respect the sizes of the WT control fruits. This is again presumably due to the leaves exporting less sugar to the fruits due to the leaf senescence phenotype that the transgenic lines exhibited. If the fruits receive less sugars they would not contain as much carbon as the controls and would not grow so large.

I also found that there was a significant (P= 0.01) reduction in all of the transgenic lines in comparison with the WT control with respect to both average number of fruit per plant and fruit set. This is despite the increased numbers of flowers in all of the transgenic lines in comparison with the WT control. The factors that control fruit set are not well understood, but it is again reasonable to assume that supply of sugars from the leaves would be important. There would be a selective advantage for the plant if a reduced supply of carbon were distributed between a smaller numbers of fruits than normal. These fruits would be larger and contain more nutrients than they would otherwise have done and would, therefore, have an increased chance of producing viable seeds than smaller fruits containing fewer nutrients.

The problem with all the data presented in this chapter was that there was a completely unexpected and drastic effect on leaf growth and photosynthesis caused by the transformation. This means that it is impossible to separate out phenotypes caused by reduction of the GWD protein in the leaf to those caused by reductions in the fruits. The original aim of this experiment, for example, was to try and delay starch degradation in the fruits through repression of the GWD protein, but this was something that turned out not to be feasible with the fruits accumulating actually less starch in the transgenic lines. In a previous chapter I used a fruit specific promoter to reduce the activity of cp-FBPase and it would be possible to repress the GWD protein also in a fruit specific manner using this promoter. Production of such plants should enable the elucidation of the influence of the GWD protein on fruit metabolism.


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From the data presented in this chapter it can be concluded that: (A) The GWD protein is mainly present in green, but not red tomato fruit. (B) Repression of the GWD proteins in tomato plants leads to early senescence of leaves presumably with a concomitant reduction in photosynthesis. (C) The leaf senescence phenotype leads to large alterations of metabolism in both leaves and fruits.

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Summary

Carbohydrate metabolism was studied during the development of fruits of the tomato cultivar Micro-Tom. The metabolism of the pericarp and placental tissues was found to be different. Starch being degraded more slowly in the placenta than in the pericarp, while soluble sugars accumulated to a greater extent in the pericarp. The activities of glycolytic enzymes tended to peak at 40 DAF. Two of these, phosphoenolpyruvate phosphatase and pyruvate kinase, showed a dramatic increase in activity just before this peak possibly indicating a role in up-regulating glycolysis to generate ATP for climacteric respiration. The expression of some plastidial transporters was also studied. Both the triose phosphate transporter (TPT) and Glc-6-P transporter was expressed greatest in green fruits, before declining. The expression of the triose phosphate transporter (TPT) was greater than that of Glc-6-P transporter. The ATP/ADP transporter was expressed to a low level throughout fruit development. These changes in transcript profiles are reflective of a switch from partially photosynthetic to fully heterotrophic metabolism. Whilst these characteristics are largely equivalent to those previously observed for normal sized tomato cultivars and as such indicated the suitability of Micro-Tom for studies of carbohydrate metabolism repeated failure to transform this cultivar made it inappropriate for further study.

Activity repression using potato cDNA encoding for the cp-FBPase, AGPase, and the GWD-protein for antisense inhibition studies was therefore performed in normal sized tomatoes of the cultivar Moneymaker. In the case of cp-FBPase, transgenic plants were isolated in which this activity was reduced by more 50% of the WT control in green fruits. Immunoblots indicated that the chloroplastidial isoform was almost completely eliminated in the most strongly inhibited lines. Measurements of metabolite levels in green fruits of the transgenic plants were consistent with an inhibition of photosynthesis, but there was little differences in the levels of metabolites or of other key enzyme activities at other time points. Consistent with the inhibition in photosynthesis the average weight and size of fully ripe fruits were significantly decreased by up to 20% in the transgenic lines. In addition the fruit set in these plants was markedly reduced, however from the present study it was not able to discriminate the reason for this.

In the case of AGPase, transgenic plants were isolated in which this activity was reduced by more 90% of the control in green fruits with immunoblots indicating that the AGPase was almost completely eliminated in the strongly inhibited line. Analysis of metabolites through


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development revealed little change in early development but a decreased content of glucose and fructose at latter stages of development. Furthermore, the line exhibiting the greatest level of AGPase inhibition was characterised by a depressed starch content. Phosphorylated intermediates determined in green fruit were also largely unchanged with the exception that 3-PGA and PEP which were significantly decreased in the strongly inhibited line. The AGPase antisense plants were characterised by significant reduction in fruit yield and the strongest line also exhibited a delayed flowering, however, from this study it was not able to explain why this phenomenon appears.

In the case of GWD protein, transgenic plants were selected by immunoblots in leaves which revealed that the GWD protein was almost completely eliminated in all transgenic lines (further experiments confirmed this was also true in the pericarp of the transgenics). Western blot analysis of GWD protein abundance revealed that it was present in green but not red fruit in the WT control. GWD-transgenic plants were phenotypically dramatically different from wild WT control, where, leaves of these plants senesced much earlier than the WT control. Analysis of metabolites through development revealed large change in early development (with respect starch and fructose content) but a decreased content of glucose and fructose at latter stages of development. On the other hand, sucrose concentration was low, and was decreased in GWD transgenic lines through development. Analysis of leaf metabolites revealed that glucose and sucrose and starch concentrations were increased in leaves in the transgenic lines, but fructose concentration was significantly decreased in leaves in the transgenic lines. The average weight and size of fully ripe fruits were high significantly decreased by up to 33% and 15% in all transgenic lines in comparison with the WT control with respect to average of weight and size respectively. Furthermore, the time of flowering was significantly delayed in these lines and the fruit set was dramatically reduced. However, the large changes in leaf metabolism combined with the fact that these are opposite in trend to those observed in the fruit make it hard to dissect the role of GWD protein in the fruit and suggests that the use of a fruit specific promoter have been a better approach by which to address this question.

The role of three enzymes (cp-FBPase, AGPase and GWD protein) are thought to influence the accumulation of starch in early development in tomato fruit were studied using antisense technique under the control of the patatin B33 promoter in the case of cp-FBPase, and the CaMV 35S promoter in the case of AGPase and GWD protein. It appears that repression of cp-FBPase and AGPase in tomato fruits does not influence metabolite levels as greatly as it


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does in leaves, possibly because any alterations are buffered by the ability of the fruit to import sugars. On the other hand, the repression of GWD protein in tomato fruits has been strongly affected on metabolite levels.


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Zusammenfassung

Während der Entwicklung von Früchten der Tomate (Sorte „Micro-Tom“) wurde der Kohlenhydrat-Stoffwechsel untersucht. Es wurde ein Unterschied zwischen dem Metabolismus im Perikarp und dem des Plazenta-Gewebes gefunden. Stärke wurde in der Plazenta langsamer abgebaut als im Perikarp, während lösliche Zucker im Perikarp stärker akkumulierten. Die Aktivitäten der glykolytischen Enzyme tendierten zu einem Maximum 40 Tage nach der Blüte. Zwei davon, Phosphoenolpyruvat-Phosphatase und Pyruvat-Kinase, zeigten einen starken Anstieg der Aktivität kurz vor diesem Maximum. Diese Tatsache weist möglicherweise auf eine Rolle dieser Enzyme in der Hochregulierung der Glykolyse, um ATP für die klimakterische Respiration zu erzeugen, hin. Weiterhin wurde die Expression einiger plastidärer Transporter untersucht. Sowohl der Triosephosphat-Tranporter (TPT) als auch der Glukose-6-phosphat-Transporter wurde am stärksten in grünen Früchten exprimiert, danach nahm die Expression ab. Der ATP/ADP-Transporter wurde während der Fruchtentwicklung nur schwach exprimiert. Diese Änderungen der Transkriptionsprofile deuten auf einen Wechsel von teilweise photosynthetischem zu vollständig heterotrophem Metabolismus hin. Diese Eigenschaften entsprechen zwar weitgehend den vorher in normalgroßen Tomaten-Sorten beobachteten und schlagen dadurch die Eignung der Sorte „Micro-Tom“ für das Studium des Kohlenhydrat-Stoffwechsels vor; jedoch erwies sich diese Sorte letztendlich als ungeeignet, da mehrere Versuche einer Transformation erfolglos blieben.

Stattdessen wurde die Sorte „Moneymaker“ mit normalgroßen Früchten für Untersuchungen zur Repression der Aktivität von plastidärer FBPase, AGPase und GWD-Protein mittels Antisense-Inhibition verwendet. Im Falle der plastidären FBPase wurden transgene Pflanzen isoliert, in denen diese Aktivität in grünen Früchten um mehr als 50% im Vergleich zur Wildtyp-Kontrolle reduziert war. Ein Immunoblot zeigte, daß die plastidäre Isoform in den am stärksten inhibierten Linien fast nicht mehr vorhanden war. Die Messungen verschiedener Metaboliten-Konzentrationen in grünen Früchten der transgenen Pflanzen waren zwar im Einklang mit einer Inhibierung der Photosynthese, aber es konnten kaum Unterschiede der Metaboliten-Konzentrationen oder der Aktivitäten von Schlüssel-Enzymen für andere Zeitpunkte in der Fruchtentwicklung gefunden werden. Entsprechend der Inhibierung der Photosynthese war das durchschnittliche Gewicht und die Größe vollreifer Früchte in den transgenen Linien signifikant (um bis zu 20%) kleiner als im Wildtyp.


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Desweiteren war die Fruchtanlage in diesen Pflanzen deutlich reduziert, es war jedoch in der vorliegenden Studie nicht möglich, einen Grund für diese Reduktion zu finden.

Im Falle der AGPase wurden transgene Pflanzen isoliert, in denen diese Aktivität in grünen Früchten um mehr als 90% im Vergleich zur Wildtyp-Kontrolle reduziert war. Auch hier zeigte ein Immunoblot, daß die plastidäre Isoform in der am stärksten inhibierten Linie fast nicht mehr vorhanden war. Die Analyse der Metaboliten während der Entwicklung der Frucht zeigte nur geringe Änderungen in der frühen Entwicklung, jedoch eine geringere Glukose- und Fructose-Konzentration in späteren Stadien der Entwicklung. Desweiteren zeigte die Linie mit der stärksten AGPase-Inhibition einen verminderten Stärkegehalt. Phosphorylierte Zwischenprodukte in grünen Früchten waren auch weitgehend unverändert, mit Ausnahme von 3-PGA und PEP, die in der am stärksten inhibierten Linie deutlich abnahmen. Die AGPase-Antisense Pflanzen zeigten eine erhebliche Abnahme der Fruchtausbeute, und die stärkste Linie wurde ausserdem durch ein verspätetes Blühen charakterisiert. Es war jedoch in dieser Studie nicht möglich herauszufinden, warum es zu diesen Phänomenen kommt.

Im Falle des GWD Proteins wurden transgene Pflanzen durch Immunoblots mit Blättern isoliert. Die Blots zeigten, daß das GWD Protein in allen transgenen Linien fast vollständig verschwunden war (weitere Experimente zeigten, daß dies auch im Perikarp der transgenen Früchte der Fall war). Western-Blot-Analysen der Verbreitung des GWD Proteins zeigten, daß dieses in grünen, nicht aber in roten Früchten des Wildtyp vorkommt. Die transgenen GWD Protein Pflanzen zeigten eine drastische phänotypische Veränderung im Vergleich zum Wildtyp. Die Blätter dieser Pflanzen wiesen eine extrem frühe Seneszenz auf. Eine Analyse der Metaboliten während der Entwicklung zeigte große Veränderungen in den frühen Entwicklungsstadien der Frucht (bezüglich Stärke- und Fructosegehalt), aber einen verringerten Gehalt an Glukose und Fruktose in späteren Entwicklungsstadien. Auf der anderen Seite war die Saccharosekonzentration gering, und nahm in den GWD Pflanzen während der Entwicklung ab. Eine Analyse der Metaboliten im Blatt brachte hervor, daß die Glukose-, Saccharose- und Stärkekonzentration in den transgenen Pflanzen im Vergleich zum Wildtyp erhöht war, die Fruktose-Konzentration hingegen war in Blättern deutlich geringer in den transgenen Linien. Gewicht und Größe der vollreifen Früchte waren im Durchschnitt um bis zu 33% bzw. 15% im Vergleich zu der Wildtyp-Kontrolle erhöht. Desweiteren war der Blütezeitpunkt in diesen Linien deutlich verspätet und die Fruchtmenge war sehr stark reduziert. Die starken Änderungen des Metabolismus im Blatt zusammen mit der Tatsache,


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daß diese einen gegenläufigen Trend zu denen der Frucht aufweisen, erschweren allerdings den Schluß auf eine Rolle des GWD Proteins in der Frucht. Der Gebrauch eines fruchtspezifischen Promoters wäre ein besserer Ansatz gewesen, diese Frage zu untersuchen.

Es besteht die Hypothese, daß die Rolle dieser drei Enzyme (plastidäre FBPase, AGPase und GWD Protein) eine Beeinflussung der Stärke-Akkumulation in der frühen Entwicklung der Tomaten-Frucht ist. Diese Hypothese wurde durch Antisense-Technik mit der plastidären FBPase (unter der Kontrolle des B33 Promoters), sowie mit der AGPase und dem GWD Protein (beide unter der Kontrolle des CaMV 35S-Promoters) untersucht. Die Repression von plastidärer FBPase oder AGPase in der Frucht der Tomate scheint die Metaboliten-Konzentrationen nicht so stark wie in den Blättern beobachtet zu beeinflussen. Der Grund hierfür ist wahrscheinlich, daß jede Veränderung durch die Fähigkeit der Frucht, Zucker zu importieren, abgepuffert wird. Auf der anderen Seite hatte die Repression des GWD Proteins in der Frucht der Tomate starke Effekte auf die Metaboliten-Konzentrationen.


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