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

Dissertation

zur Erlangung des akademischen Grades
doctor rerum agriculturarum
(Dr. rer. agr.)

Eingereicht an der
Landwirtschaftich-Gärtnerischen Fakultät
der Humboldt-Universität zu Berlin


vonHazem Abd El-Rahman Obiadalla Ali (M.Sc.),
geb. am 14.09.1969 in Sohag, Ägypten

Präsident der
Humboldt-Universität zu Berlin
Prof. Dr. Jürgen Mlynek

Dekan der
Landwirtschaftich-Gärtnerischen Fakultät
Prof. Dr. Uwe Jens Nagel

Gutachter:
Prof. Dr. F. Pohlheim
Prof. Dr. L. Willmitzer
Dr. J. Kossmann

Tag der mündlichen Prüfung: 10.06.2003

The work presented in this thesis was carried out between November 1999 and December 2002 at the Max-Planck-Institute für Molekulare Pflanzenphysiologie, Golm.

This Ph.D. thesis is the account of work done between November 1999 and December 2002 in the department of Prof. L. Willmitzer in the Max-Planck Institute of Molecular Plant Physiology, Golm, Germany. It is results of my own work and has not been submitted for any degree or Ph.D. at any other university.


Seiten: [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119]

Inhaltsverzeichnis

TitelseiteUnderstanding of Carbon Partitioning in Tomato Fruit
Selbständigkeitserklärung
Widmung
1 General Introduction
2 Review of literature
2.1Carbon Metabolism in Photosynthetic Tissue
2.1.1Feedforward control of photosynthesis
2.1.2Feedback control of photosynthesis
2.2Carbohydrate Allocation from photosynthetic “source“ to heterotrophic “sink Tissues“
2.3Mobilisation of Sucrose in sink tissues
2.4Uptake of carbon into amyloplasts
2.5The synthesis of starch
2.6Starch degradation
2.7Starch phosphorylation
2.8Glycolysis
2.9Fruit metabolism
3 Material and Methods
3.1Chemicals
3.2Vectors and Bacterial Strains
3.2.1Vectors
3.2.2Bacterial
3.3Transformation and Cultivation of Bacteria
3.4DNA manipulations
3.5Cloning
3.6Plant Material
3.7Sampling of fruits
3.8Transformation and Cultivation of tomato
3.9Selection of plants with reduced cp-FBPase AGPase and GWD protein
3.9.1Selection of plants with reduced cp-FBPase activity
3.9.2Selection of plants with reduced AGPase activity
3.9.3Selection of plants with reduced GWD protein levels
3.10Western Blot Analysis
3.11RNA (Northern) Blot Analysis
3.12Determination of enzyme Maximum Catalytic Activities
3.12.1Extraction Procedures and Assay Condition
3.12.2Phosphoglucoisomerase (EC 5.3.1.9)
3.12.3Phosphoglucomutase (EC 5.4.2.2)
3.12.4Hexokinase (EC 2.7.1.1)
3.12.5Fructokinase (EC 2.7.1.4)
3.12.6UDP-glucose Pyrophosphorylase (EC 2.7.7.9)
3.12.7Sucrose Synthase (EC 2.4.1.13)
3.12.8Enolase (EC 4.2.1.11)
3.12.9Triose Phosphate Isomerase (EC 5.3.1.1)
3.12.10Phosphoglycerate Kinase (EC 2.7.2.3)
3.12.11Phosphofructokinase (EC 2.7.1.11)
3.12.12Pyruphosphate dependent Phosphofructokinase (EC 2.7.1.90)
3.12.13Glyceraldehyde 3-Phosphate dehydrogenase (EC 1.2.1.12)
3.12.14Pyruvate Kinase (2.7.1.40)
3.12.15Phosphoenolpyruvate Phosphatase (3.1.3.60)
3.12.16Fructose-1, 6-bisphosphatase (EC 3.1.3.11)
3.12.17ADP-glucose Pyrophosphorylase (EC 2.2.7.27)
3.12.18Acid Invertase (EC 3.2.1.26)
3.13Determination of Soluble Sugars and Starch Content
3.14Determination of Metabolic Intermediates
3.15Analysis of fruit yield and flowers
3.15.1Analysis of fruit weight
3.15.2Analysis of fruit size
3.15.3Fruit setting
3.15.4Date of 50% flowering
3.16Statistical Analysis of Data
4 Analysis of Carbohydrate Metabolism in Micro-Tom Fruits
4.1Introduction
4.2Aim of the work
4.3Results
4.3.1Development of fruit of tomato cultivar Micro-Tom
4.3.2Starch and soluble sugars in developing fruits of Micro-Tom
4.3.3Changes in activities in enzymes involved in conversion of sucrose to starch
4.3.4Changes in activities in enzymes involved in glycolysis or the Calvin cycle
4.3.5RNA blots of plastidial transporters
4.4Discussion and conclusion
5 Analysis of the Function of Chloroplastic Fructose 1,6-bisphosphatase in Tomato Fruit
5.1Introduction
5.2Aim of the work
5.3Results
5.3.1Recovery of Plants with Reduced FBPase Activity in the Pericarp of Tomato Fruit.
5.3.2Starch and soluble sugar contents in the pericarp of the WT and transgenic lines
5.3.3Changes in activities in enzymes involved in conversion of sucrose to starch
5.3.4Concentration of Metabolic Intermediates in the pericarp of the WT control and transgenic lines
5.3.5Analysis of fruit yield
5.3.6Number of flower, fruit per plant, fruit set and number of days to 50% flowering.
5.4Discussion and conclusion
6 Functional Analysis of ADP-glucose Pyrophosphorylase in Tomato Fruit
6.1Introduction
6.2Aim of the work
6.3Results
6.3.1Recovery of plants with reduced AGPase activity in the pericarp of tomato fruit
6.3.2Starch and soluble sugar contents in the pericarp of the WT and transgenic lines
6.3.3Changes in activities in enzymes involved in conversion of sucrose to starch
6.3.4Concentration of metabolic intermediates in the pericarp of the WT control and transgenic lines
6.3.5Analysis of fruit yield
6.3.6Number of flowers, fruits per plant, fruit set and number of days to 50% flowering
6.4Discussion and conclusion
7 Analysis of the Function of the GWD protein in Tomato Fruit
7.1Introduction
7.2Aim of the work
7.3Results
7.3.1Recovery of Tomato Plants with Repression of the GWD Protein
7.3.2Starch and soluble sugar contents in the pericarp of the WT and transgenic lines
7.3.3Starch and soluble sugar contents in the leaves of the WT and transgenic lines
7.3.4Changes in activities in enzymes involved in conversion of sucrose to starch
7.3.5Analysis of fruit yield
7.3.6Number of flower, fruit per plant, fruit set and number of days to 50% flowering
7.4Discussion and conclusions
Bibliographie Literature Cited
Danksagung
Lebenslauf

Tabellenverzeichnis

Table 1: Metabolite concentrations in the pericarp of 30 DAF old WT control and alphacp-FBP-transgenic fruits.
Table 2: Weights and sizes of ripe tomato fruits in the WT control and alphacp-FBP-transgenic lines.
Table 3: Number of flowers, fruits, fruit set and number of days to 50% flowering in the WT control and alphacp-FBP-transgenic lines.
Table 4: Metabolite concentrations in the pericarp of 30 DAF old WT control and alpha-AGP-transgenic lines.
Table 5: Weights and sizes of ripe tomato fruits in the WT control and alpha-AGP-transgenic lines.
Table 6: Number of flowers, fruits, fruit set and number of days to 50% flowering in the WT control and the transgenic lines.
Table 7: Weights and sizes of ripe tomato fruits in WT control and alpha-GWD-transgenic lines.
Table. 8: Number of flowers, fruit set and number of days to 50% flowering in the WT control and alpha-GWD-transgenic lines.

Abbildungsverzeichnis

Figure 1: The role of Fru-2,6-P2 in feedforward control of sucrose synthesis.
Figure 2: The role of Fru-2,6-P2 in feedback control of sucrose synthesis.
Figure 3: The predominant route of sucrose unloading and subsequent mobilization.
Figure 4: Developmental series of tomato fruits from Micro-Tom cultivar.
Figure 5: Starch and soluble sugar contents in pericarp and placental tissues of tomato cultivar Micro-Tom during development. (A) Starch. (B) Sucrose. (C) Fructose. (D) Glucose. Data represent the mean of five independent measurements + SE.
Figure 6: Activities of enzymes involved in the conversion of sucrose to starch in the pericarp and placental tissues of fruit of the tomato cultivar Micro-Tom. (A) SuSy. (B) Acid invertase. (C) UDPase. (D) PGM. (E) AGPase. Data represent the mean of five independent measurements + SE.
Figure 7: Activities of some glycolytic and clavin cycle enzymes in pericarp and placental tissues of fruit from the tomato cultivar Micro-Tom during its development.(A) HK. (B) FK. (C) FGI. (D) FBPase. (E) PPi-PFK. (F) PFK. (G) TPI. (H) G3P DH. (I) PGK. (J) Enolase. (K) PK. (L) PEP phosphatase. Data represent the mean of five independent measurements+ SE.
Figure 8: RNA blot analysis of some plastidial transporters throughout fruit development in the tomato cultivar Micro-Tom. TPT in (A) pericarp and (B) placental tissues. Glc-6-P transporter in (C) pericarp and (D) placental tissues. ATP/ADP transporter in (E) pericarp and (F) placental tissues.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Figure 18: Some 65 DAF old fruits from alpha-AGP-transgenic line #7 (bottom) in comparison with the WT control fruit (above).
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.
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].
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
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.
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.
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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