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

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Kapitel 6. Functional Analysis of ADP-glucose Pyrophosphorylase in Tomato Fruit

6.1 Introduction

ADP-glucose pyrophosphorylase (AGPase) catalyses the first reaction on the committed pathway of starch biosynthesis. In higher plants, AGPase is a heterotetramer consisting of two large and two small sub-units which are 54 and 51 kDa in size respectively (see Preiss, 1991). It is known that it is essential for starch production as reductions in its activity in mutant and transgenic plants leads to reductions in starch contents (Tsai and Nelson 1966, Dickinson and Preiss 1969; Lin et al., 1988; Smith et al., 1989; Müller-Röber et al., 1992).

Until recently it was thought that the AGPase enzyme was located solely in the plastid in all plant species. Evidence over the past decade, however, has indicated that in cereal endosperm there is a second isoform present in the cytosol also, which contributes the majority of the total activity, at least in barley, maize, wheat and rice (Thorbjørnsen et al, 1996a, Denyer et al. 1996; Sikka et al., 2001; Burton et al., 2002). As starch is manufactured within the plastid this would mean that in cereal endosperm any ADP-glucose produced in the cytosol would have to be imported into the amyloplast. It is thought that this is performed by a protein named Brittle-1. Mutations in the gene coding for this protein in maize lead to a decreased starch content (Sullivan et al., 1991) and increased ADP-glucose concentrations (Shannon et al., 1996), both of which would be expected if the protein has this function. In addition, it has been localized in the amyloplast membrane (Cao et al., 1995, Sullivan and Kaneko, 1995) and analysis of its primary protein sequence indicates that it is a sugar-nucleotide transporter. There is still some controversy about whether a similar system is present in tomato fruits. One immunolocalisation study indicated that AGPase was present both inside and outside the plastid in tomato pericarp (Chen et al., 1998), however, its activity was found only in the plastid fraction upon sub-cellular fractionation (Beckles et al., 2001a).

Starch accumulates transiently in tomato fruits being present when they are immature, but not when they are ripe. Its role in tomato fruit metabolism is not understood, but it has been proposed that carbohydrate metabolism in this organ is controlled by a number of different futile cycles, one of which involves continuous synthesis and degradation of starch (Nguyen-Quoc and Foyer, 2001). In that paper it was proposed that repression of AGPase in tomato fruits would be a good method to examine any such futile cycle.


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6.2 Aim of the work

The aim of the work described in this chapter is to repress the activity of AGPase in tomato plants and study the effect on fruit metabolism. It would be hoped that these data would help to answer the question as to whether AGPase is situated both inside and outside the plastid in tomato fruit, and to help to understand the role, if any, of a futile cycle of starch degradation and synthesis in carbohydrate metabolism in tomato fruits.

6.3 Results

6.3.1 Recovery of plants with reduced AGPase activity in the pericarp of tomato fruit

The pericarp of 25 days after flowering (DAF) old tomato fruits were analysed for AGPase activity in forty transgenic lines. Three lines (#2, #7 and #11) showed reductions in AGPase activity and were chosen for further study (see Material and Methods). The transgenic plants themselves were phenotypically identical to the untransformed control (Fig. 14).

AGPase activity was studied throughout fruit development in the pericarp of these plants. There was a significant reduction in AGPase activity in all three transgenic lines throughout fruit developing in comparison with the WT control (Fig. 15A). Initially the reduction in activity of line #7 was 90%, and in the other two lines 70%. The activity in the control decreased during the ripening process, but the activity in all transgenic lines relatively constant during the ripening process. AGPase activities were significantly reduced in comparison with the WT control until 55 DAF in transgenic line #2, and at every time point studied in the other two lines.

Immunoblots using an antibody raised against the a sub-unit of maize AGPase (Müller-Röber, et al., 1992), but which recognizes the tomato protein also indicated that it was completely eliminated in 25 DAF fruit of the lines #7 and greatly reduced in the lines #11 and #2 (Fig. 15B).

6.3.2 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. 16A). In the WT control the concentration was approximately 18µmol hexose (g FW)-1 at 25 DAF, and was 0.3µmol hexose (g FW)-1 at the final sample point. The starch contents in lines #2 and #11 were not significantly different in comparison with the control over the entire sampling period, but


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those of line #7 were significantly reduced until 50 DAF, after which there were no differences.

Figure 14: Aerial parts of plants in both WT control and alpha-AGP-transgenic lines after 13 weeks growth in the glasshouse. From left to right: WT control, transgenic line #2, transgenic line #7, transgenic line #11 and transgenic line #11. The alpha-AGP plants are phenotypically identical to the untransformed WT control.

Both glucose and fructose concentrations increased slightly over time in both the control and the transgenic lines. Glucose concentrations were significantly increased in the WT control in comparison with the three transgenic lines, but only between 55 and 70 DAF. At these time points the concentrations in the WT control fruits increased from approximately 70 to 110 µmol (g FW)-1, while in the transgenic fruits they increased from approximately 60 to 90 µmol hexose (g FW)-1 (Fig. 16B). There were no significant differences in fructose concentrations between the transgenic lines and the WT control (Fig. 16C).

Sucrose concentrations stayed between 2-4 µmol (g FW)-1 in all lines from the first sampling point until 60 DAF, after this point they increased to between 6-8 µmol (g FW)-1. The only significant differences in sucrose concentrations between the transgenic lines and the WT


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control were at 65 and 70 DAF (Fig. 16D). At these time points there was a significantly greater concentration of sucrose in the pericarp from control than the transgenics.

Figure 15: AGPase activity during developmental stage (A) and Western blot analysis in green (25 DAF) (B) in the pericarp of WT control and alpha-AGP-transgenic lines. Total soluble fruit protein (25µg) was subjected to SDS-PAGE on a 10% (w/v) gel.

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

Sucrose synthase (SuSy) activity approximately 100nmol min-1 (mg protein)-1 in the pericarp of the control plants at 25 DAF. This decreased to about 25nmol min-1 (mg protein)-1 at 45 DAF, after which it remained relatively constant. There were no significant differences in


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SuSy activity between the transgenic lines and the WT control except at 45 DAF in line #7 where the activity was significantly greater than in the WT control (Fig 17A).

Figure 16: Starch and soluble sugar contents in the pericarp of the WT control and alpha-AGP-transgenic lines.(A) Starch. (B) Glucose. (C) Fructose. (D) Sucrose. Data represent the mean of five independent measurements + SE in both WT control and transgenic line #7, but four independent measurements + SE in transgenic line #2 and transgenic line #11.


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The activity of UDP-glucose pyrophosphorylase (UGPase) increased from approximately 500nmol min-1 (mg protein)-1 in the WT control at 25 DAF to 1000nmol min-1 (mg protein)-1 at 55 DAF, before decreasing to 530nmol min-1 (mg protein)-1 at 65DAF. Some significant differences in activities were noted in the transgenic lines. Activities were significantly reduced in line #2 at 35 DAF and in line #7 at 65 DAF, however the activitiy was significantly increased in line #11 at 45 DAF (Fig 17B).

Phosphoglucomutase (PGM) activity increased from 416nmol min-1 (mg protein)-1 to 732nmol min-1 (mg protein)-1 in pericarp from the control between 25 and 45 DAF. It then decreased to 306nmol min-1 (mg protein)-1 at 65 DAF. The activity in all the transgenic lines was significantly reduced in comparison with the control at 35DAF and was also significantly reduced in line #2 at 45DAF (Fig 17C).

Fructose-1,6-bisphosphatase (FBPase) activity decreased from 12nmol min-1 (mg protein)-1 at 25 DAF to 4nmol min-1 (mg protein)-1 at 65 DAF in the WT control. The activities in the transgenic lines were not significantly altered, except at 65 DAF in line #7 when a significant reduction in activity was found (Fig 17D).

6.3.4 Concentration of metabolic intermediates in the pericarp of the WT control and transgenic lines

The concentration of some metabolites were measured in trichloroacetic acid extracts from 30 DAF fruits. The metabolites determined were Glc-6-P, Glc-1-P, Fru-6-P, 3-PGA, PEP, pyruvate and Pi. The data are representing in Table 4. Few significant differences were found between the transgenic lines and the WT control. Both PEP and 3-PGA were significantly reduced (P= 0.01, P= 0.05 respectively) in line #7. Pyruvate concentrations were also significantly reduced (P= 0.05) in lines #2 and #11. No significant differences were found between the transgenic lines and the control for total hexose-P, the hexose-P to 3-PGA ratio and the total phosphateester to Pi ratio, however the 3-PGA to Pi ratio was significantly reduced (P= 0.01) in line #7.

6.3.5 Analysis of fruit yield

Fruit were harvested, and their weights and sizes determined after 65 DAF. Some fruits of one transgenic line (#7) can be seen in comparison with the WT control in Fig. 18. As can be seen in Table 5 in one transgenic line (#7) the average weights and sizes of harvested fruits were


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significantly (P= 0.01) reduced in comparison with the WT control. This reduction was 22% and 10% of respectively the weights and sizes of the WT control fruits.

Figure 17: Activities of enzymes involved in the conversion of sucrose to starch in pericarp of the WT control and alpha-AGP transgenic lines of fruit of the tomato cultivar Moneymaker. (A) SuSy. (B) UGPase. (C) PGM. (D) FBPase. Data represent the mean of five independent measurements + SE in both WT control and transgenic line #7 and four independent measurements + SE in transgenic line #2 and transgenic line #11.


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Table 4: Metabolite concentrations in the pericarp of 30 DAF old WT control and alpha-AGP-transgenic lines.

Conc. nmol.(g FW)-1

Lines

WT

#2

#7

#11

G6P

67.6 + 5.5

64.9 + 3.9

65.9 + 3.0

56.1 + 1.8

G1P

9.3 + 0.7

8.5 + 0.4

7.9 + 0.3

8.6 + 0.1

F6P

22.0 + 2.1

21.2 + 2.3

21.0 + 1.1

18.1 + 1.2

Total Hexose-P

98.8 + 7.8

94.6 + 6.0

94.8 + 4.1

83.8 + 2.6

3-PGA

17.4 + 0.6

16.4 + 1.7

13.8 + 0.8

18.7 + 0.5

PEP

5.3 + 0.6

4.1 + 0.5

2.4 + 0.4

5.0 + 0.4

Pyruvate

6.3 + 0.9

3.4 + 0.2

4.1 + 0.5

3.5 + 0.4

Pi

1.1 + 0.01

1.2 + 0.11

1.2 + 0.11

1.2 + 0.05

Ratio

 

 

 

 

Hexose-P/3-PGA

5.1 + 0.29

7.0 + 0.92

6.7 + 0.20

4.4 + 0.05

P-ester/Pi

106.0 + 7.8

93.2 + 7.5

90.2 + 4.6

88.9 + 6.0

3-PGA/Pi

15.8 + 0.6

14.1 + 2.1

11.3 + 0.4

16.2 + 1.2

The data represent means + SE of five independent samples. Significant differences (P= 0.05 and P= 0.01, Student‘s t-test) are in bold.

Table 5: Weights and sizes of ripe tomato fruits in the WT control and alpha-AGP-transgenic lines.

Lines

Average

Weight of Fruit (g)

Size of Fruit (cm)

WT

#2

#7

#11

56.7 + 2.0 (n=53)

59.3 + 2.7 (n=44)

44.7 + 1.6 (n=46)

55.6 + 1.9 (n=44)

4.8 + 0.07 (n=53)

4.9 + 0.08 (n=44)

4.3 + 0.06 (n=46)

4.8 + 0.07 (n=44)

Data are means + SE, number of sample is in parentheses. Significant differences (P= 0,01), Student‘s t-test are in bold.


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Figure 18: Some 65 DAF old fruits from alpha-AGP-transgenic line #7 (bottom) in comparison with the WT control fruit (above).

6.3.6 Number of flowers, fruits per plant, fruit set and number of days to 50% flowering

Data of these traits are presented in Table 6. The only significant difference in the transgenic lines was that line #7 took longer to reach 50% of its flowers than the control.

Table 6: Number of flowers, fruits, fruit set and number of days to 50% flowering in the WT control and the transgenic lines.

Lines

Average

No. of flower/plant

No. of fruit/plant

Fruit set %

No. of days to 50% flowering

WT

#2

#7

#11

50.8 + 4.7

49.0 + 3.6

45.4 + 3.4

56.3 + 3.0

42.2 + 3.4

39.0 + 3.7

34.2 + 1.9

48.5 + 1.9

83.6 + 3.8

79.2 + 2.3

76.6 + 6.0

86.4 + 1.3

54.0 + 0.5

55.5 + 1.2

59.8 + 1.3

56.5 + 1.0

Data represent the mean of five independent measurements + SE in the WT control and in transgenic line #7, but four independent measurements + SE in both transgenic lines #2 and #11. Significant (P= 0,01, Student‘s t-test) differences from the control are in bold.


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6.4 Discussion and conclusion

In this study I have described the production of transgenic tomato plants repressed in AGPase activity using a constitutive promoter. The transgenic plants appeared phenotypically normal, indicating that repression of AGPase activity has no effect on growth of the plants under the conditions used. In addition the activities of other enzymes involved in the conversion of sucrose to starch were not consistently altered indiacting that reductions in AGPase activity did not lead to pleiotropic effects on fruit metabolism.

AGPase activity was demonstrated to be reduced throughout fruit development in the pericarp of these plants, and the degree of reduction correlated with reductions in amount of AGPase protein as determined by immunoblots. This reduction in activity ranged between 70 to 90% of the WT control activity in the different transgenic lines at 25 DAF, with line #7 being the most repressed. AGPase activity in the WT control decreased during fruit ripening, as did starch contents in the pericarp. This is interesting as AGPase activity has often been associated with starch accumulation both in tomato (Yelle et al., 1988) and other plants (Okita, 1992; Preiss, 1988, 1991). It has indeed been suggested that it may catalyse a rate-limiting step in starch accumulation (Stark et al., 1992), although this still remains a controversial idea. My data indicates that AGPase activity is in excess in tomato fruits as repression of its activity by 70% did not greatly alter starch accumulation. It was only in the one line (#7) where activity was reduced by 90 % that an effect on starch accumulation was found. The starch content of that line was only approximately 25 % that of the WT control. This is similar to the data found in the study of Müller-Röber et al. (1992), who repressed AGPase activity in potatoes using the same construct as in this study. They found that decreased starch contents were only found when AGPase activity was reduced by more than 50 %. This indicates that in tomato fruit AGPase does not control the amount of starch that accumulates. If that were the case it would be expected that a straight-line relationship would be found between its activity and starch content. Although there is obviously some influence of AGPase on starch content (as demonstrated by line #7), the reductions in activities in lines #2 and #11 were not mirrored by reductions in its accumulation. This indicates that other enzymes also influence the rate of its synthesis. In Arabidopsis and potato it has been demonstrated that plastidial PGM - which catalyses the step prior to AGPase - can influence the rate of starch synthesis (Neuhaus and Stitt, 1990; Fernie et al., 2001), and it might be that this is the case in tomato also.


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In a previous study AGPase was repressed in potato (Müller-Röber et al., 1992), which led to plants producing tubers containing only a small amount of starch, but high levels of soluble sugars, mainly sucrose and glucose. Such drastic differences were not found in the fruits of the transgenic tomato plants in this study. As was said above, starch contents were reduced in only one of the transgenic tomato lines, but this was not accompanied by increases in soluble sugar concentrations. The only differences noted in respect to this was that there were slight decreases in glucose and sucrose concentrations at the final two sampling points, a time when the fruits are ripe. These were precisely the points where it would not be expected that AGPase would have an influence as both its activity, and starch contents in the pericarp are extremely low. In addition the same reductions in glucose and sucrose were noted in the two transgenic lines where starch was not reduced (#2 and #11). Soluble sugar concentrations are often quite variable, and I feel that it is most likely that these small differences are due to the small number of probes taken (four or five) rather than due to some difference caused by the transgene.

The lack of increase in soluble sugars demonstrates that starch is not used as a major carbon reserve in tomatoes. This is again different to the situation in a potato tuber where the starch content is generally between 25-50 times greater than the maximum amount found in the pericarp in this study. Tomato fruits obviously store more carbon in the form of soluble sugars than starch. In this study in green fruits - the time point when starch contents are maximal and sugar contents minimal - there was approximately six fold more carbon present as glucose, sucrose and fructose than starch. It is, therefore, not surprising that reductions in starch in line #7 did not greatly influence sugar contents as it makes up such a small proportion of the total metabolisable carbon in the fruit.

One aim of this study was to use a genetic approach to examine whether AGPase is present solely in the plastid in tomato fruit. The reason why that is possible is that it is known that the construct I used represses a plastidial isoform of AGPase. This is demonstrated as starch contents are reduced in potato leaves where this isoform has been repressed (Leidreiter et al., 1995). Although it is now generally accepted that cereal endosperms contain an extra-plastidic AGPase isoform, it is still assumed that leaves do not as the starch is manufactured from carbon derived directly from photosynthesis, a process that occurs solely in the chloroplast. My data does not rule out the possibility of a cytosolic form of AGPase in tomato fruit as I was only able to repress up to 90 % of the AGPase activity and it is possible that the remaining 10 % of the activity is extra-plastidial, however it does demonstrate that the


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majority of the activity is in the plastid. The assumption for this statement is that there is no differential splicing mechanism of the AGPase gene in tomato. In wheat and barley it has been demonstrated that the gene coding for the small sub-unit of AGPase is subject to alternative splicing giving rise to two forms of the protein, one of which can be imported into plastids and one which cannot (Thorbjonsen et al., 1996b; Burton et al., 2002). This seems unlikely in tomato based on the strong evidence in potato that the AGPase small sub-unit gene is not differentially spliced. As potato and tomato are so closely related it is reasonable to assume that is also the case in tomato. The evidence in potato that there is no cytoplasmic AGPase is that when a bacterial form of AGPase was expressed in either the cytoplasm or plastid in potato tubers, increases in starch contents were found only upon expression in the the plastid (Stark et al., 1992). If there was a cytosolic form it would be expected that expression in the cytoplasm would lead to increased manufacture of starch also.

In this study I analysed the yield of tomato fruit and found that the both average weights and sizes of fruits in one transgenic line (#7) were significantly (P= 0.01) reduced in comparison with the WT control. This was the strongest inhibited line, and the only one with a reduced starch content, indicating that this phenotype may well be due to the inhibition of AGPase activity. Unfortunately as this was only found in one line, somaclonal variation cannot be ruled out. The same argument holds true for the delay in flowering noted in line #7. To study whether this was really an effect of strong reductions in AGPase activity, other lines which are equally strongly inhibited would have to be identified.

From the data presented in this chapter it can be concluded that: The line exhibiting the greatest level of AGPase inhibition was characterised by a depressed starch content, significant reduction in fruit yield and a delayed flowering.


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