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

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Kapitel 3. Material and Methods

3.1 Chemicals

General chemicals were obtained from Boehringer Mannheim (Mannheim), Sigma Chemical company (St.Louis, Missouri, USA) or Merck (Darmstadt).

Bactotrypton (= Select Peptone 140), Select Yeast Extract, Meat Peptone, and Select Agar were obtained from GibcoBRL Life Technologies GmbH (Paisley, Scotland, UK). Antibiotics were purchased from Sigma Chemical company (St. Louis, Missouri, USA) or Boehringer Mannheim (Mannheim) except for Betabactyl® (Smithkline Beecham Pharma, Munich).

Restriction enzymes and buffers were obtained either from Boehringer Mannheim (Mannheim) or from New England Biolabs (Beverly, Massachusetts, USA), Ready-to-GoTM T4-DNA-Ligase was brought from Pharmacia Biotech (Freiburg).

[á-32P]dCTP (110 TBq mmol-1) were purchased from Amersham Buchler (Braunschweig, Germany)

The starch determination kit (UV method; Cat. No. 207 748) and, except where noted otherwise, all biochemical enzyme purchased from Boehringer Mannheim (Mannheim).

Adenine and uridine nucleotides, NAD+, NADH, NADP+, fructose 6-phosphate, glucose 6-phosphate, glucose 1-phosphate, phosphoenolpyruvate and 3-phosphoglycerate were obtained from Boehringer Mannheim (Mannheim). All other substrates were purchased from Sigma Chemical company (St. Louis, Missouri, USA).

Rainbow TM coloured protein molecular weight marker (14 300-220 000 Da) was purchased from Amersham Buchler (Braunschweig), all other chemical for PAGE and protein determination were obtained from BioRad (Richmond, California, USA).

The peptide antibody recognising the GWD protein was kindly provided by Dr. James Lloyd Plant Research Department, Risø National Laboratory, DK-4000 Roskilde, Denmark.

3.2 Vectors and Bacterial Strains

3.2.1 Vectors

pBluescript II SK+/-

Stratagene, La Jolla, CA, USA.

pBluescript II KS+/-

Stratagene, La Jolla, CA, USA.

pBinAR

(Höfgen and Willmitzer, 1990).


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3.2.2 Bacterial

Strains Escherichia coli

Xl-1 blue

Startagene, La Jolla, CA, USA (Bullock et al., 1987).

DH5alpha

Gibco BRL, Gaithersburg, USA (Raleigh et al., 1989).

Strains Agrobacterium tumefaciens

GV2260

(Deblaere et al., 1985).

GV3101

(Koncz und Schell, 1986).

pBluescript Plasmids which include fragment cDNA‘s encoding for FBPase, AGPase and GWD were kindly provided by Dr. Jens Koßmann, Max-Planck Institute of Molecular Plant Physiology, Golm.

Aqueous plasmid stocks were kept at -20 °C prior to use. Bacterial glycerol stocks were generated as described Sambrook et al., 1989 and stored at -80°C.

3.3 Transformation and Cultivation of Bacteria

Competent E.coli XL1 Blue cells were prepared and transformed by heat-shock as described by Hanahan (1983). The cells were grown at 37°C on YT-medium plus appropriate selective antibiotic as described by Sambrook et al., (1989).

Competent Agrobacterium Tumifaciens cells were prepared according to Höfgen and Willmitzer (1990) and transformed by electroporation according to Miller et al., (1988). The cells were grown at 28°C on YEP-medium plus appropriate selective antibiotic according to Vervliet et al., (1975).

3.4 DNA manipulations

DNA manipulations were performed essentially as described by Sambrook et al., 1989. For construction of the cp-FBPase antisense gene, cDNA from potato (Kossmann et al., 1992) was digested with the restriction enzymes ASP718 and BamH1 resulting in two DNA fragments, one of approximately 800bp ant the other of 400bp. The 800bp fragment was isolated from an agarose gel using a commercially available kit (Qiagen), and was ligated in antisense orientation with respect to the patatin B33 promoter (Rocha-Sosa et al., 1989) in the ASP718/BamH1 sites of the plant transformation vector pBinARB33 producing the vector pBinB33cp-FBPase.

For construction of the AGPase antisense gene, cDNA from potato (Müller-Röber et al., 1990) was digested with the restriction enzymes EcoR1 and SmaI resulting one DNA


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fragment approximately 1.6kp. The 1.6kp fragment was isolated from an agarose gel using a commercially available kit (Qiagen), and was ligated in antisense orientation with respect to the CaMV 35S promoter in the EcoR/SmaI sites of the plant transformation vector pBinAR35S (Höfgen and Willmitzer, 1990), producing the vector pBin35S AGPase.

For construction of the GWD antisense gene, cDNA from potato (Kossmann et al., 1991) was digested with the restriction enzymes ASP718 and BamH1 resulting one DNA fragment, approximately 1.9kp. The 1.9kp fragment was isolated from an agarose gel using a commercially available kit (Qiagen), and was ligated in antisense orientation with respect to the CaMV 35S promoter in the ASP718/BamH1 sites of the plant transformation vector pBinAR35S (Höfgen and Willmitzer, 1990), producing the vector pBin35S GWD.

3.5 Cloning

Preparation and restriction of plasmids, cloning, and gel electrophoresis were performed according to Sambrook et al., (1989). Ligations were performed using the Ready-to-GoTM T4-DNA-Ligase system (Pharmacia Biotech; Freiburg) according to the manufacturer's protocol. DNA fragment were eluted from the gel and purified using Microcon Columns (Amicon Inc.; Bevery, Massachusetts, USA) according to the manufacturer's protocol.

3.6 Plant Material

Wild-type (WT) Micro-tomato (Lycopersicon esculentum cv. Micro-Tom) seeds were a kind gift of Dr. Avraham Levy (The Weizmann Institute of Science, Rehovot, Israel), whilst seeds of wild-type Moneymaker (Lycopersicon esculentum L. cv. Moneymaker) were kindly provided by Dr. Jens Koßmann, Max-Planck Institute of Molecular Plant Physiology, Golm. Seeds were sown individually in small pot (5cm diameter) in the case of Micro-Tom and in a big pot (10 cm diameter) in the case of Moneymaker in growth chamber. After two weeks the plants were transported into a glasshouse and grown illumination (16h light: 8h dark regime (approximately 250µmol photons m-2 sec-1) at 22°C temperature (20-24°C) with a relative humidity of 60-70%. Individual flowers were tagged at anthesis to accurately follow fruit ages through development, with only ten fruits per plant being allowed to develop.

3.7 Sampling of fruits

Micro-Tom and Moneymaker fruits were harvested at five days intervals between 20-60 DAF in the case of Micro-Tom and 25-70 DAF in the case of Moneymaker, which covered the transition from green to fully ripe red fruit. Harvested fruits were cut in two parts with a


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scalpel blade and the pericarp was separated from the placental tissue. In the case of Micro-Tom the placenta was then further separated from the developing seeds and jelly, and the both pericarp and placental tissues were frozen separately in liquid nitrogen, but in the case of Moneymaker only the pericarp was immediately frozen in liqued nitrogen. All samples were kept at -80°C until use.

3.8 Transformation and Cultivation of tomato

Transformation of three antisense construct (alphacp-FBPase, alpha-AGPase and? alpha-GWD protein) has been carried out using (Lycopersicon esculentum L. cv. Moneymaker) instead of (Lycopercicon esculentum cv. Micro-tom) which can not be used for transformation of these antisense construct.

Transformation of tomato (Lycopersicon esculentum cv. Moneymaker) plants was achieved by Agrobacterium Tumifaciens mediated gene transfer following the method of Rocha-Sosa et al., (1989). The selection of transgenic plants was performed on medium containing Kanamycin (Dietze et al., 1995).

3.9 Selection of plants with reduced cp-FBPase AGPase and GWD protein

Plants were maintained in tissue culture on MS-Medium (Murashing and Skoog, 1962) containing 2% (w/v) sucrose, 0.8% (w/v) Select Agar and 125µg/ml Ticarcillin Disodium/Potassium Clavulanate (Duchefa) (Timentin) under the following conditions: 22°C, 56-70% relative humidity, 3000 Lux, and a 16h light, 8h dark regime. Regenerates were screened for expression of the transgene by determining enzyme activity in the case of alpha-cp-FBPase and, by determining enzyme activity as well as starch content in the case of alpha-AGPase and by determining western blot analysis in the case of alpha-GWD protein.

3.9.1 Selection of plants with reduced cp-FBPase activity

In order to select plants with reduced cp-FBPase activity, 25 days old green fruits were harvested from sixty independent transgenic lines growing in soil. Soluble proteins were extracted from the pericarp of all lines and FBPase activity was determined in all of them. Three lines (#19, #33 and #34) showed significant reduction in total FBPase activity and were chosen for further study. Seeds from these plants were sterilized and germinated on MS media (Murashige and Skoog, 1962) containing 50mg l-1 kanamycin. Seeds that were able to grow on this media were presumed to contain the transgene and were planted in soil for further


35

analysis. In those fruits showing reduced FBPase activity the amount of the cp-FBPase was examined using western blot analysis.

3.9.2 Selection of plants with reduced AGPase activity

In order to select plants with reduced AGPase activity, 25 days old green fruits were harvested from forty independent transgenic lines growing in soil. Soluble proteins were extracted from the pericarp of all lines and AGPase activity was determined in all of them. Three lines (#2, #7 and #11) showed reductions in AGPase activity as well as in starch content and were chosen for further study. Seeds from these plants were sterilized and germinated on MS media (Murashige and Skoog, 1962) containing 50mg l-1 kanamycin. Only seeds of transgenic lines (#7) were able to grow on this media, while seeds both lines (#2 and #11) were not able to grow in this media, therefore, both seeds from WT control and all of transgenic lines were sown directly in soil for further analysis.

3.9.3 Selection of plants with reduced GWD protein levels

In order to select plants with reduced GWD expression, plant leaves were kept for 72 hour in darkness. Leaf blades in different stages of development were collected, and de-stained in 80% (v/v) ethanol at 80°C. After chlorophyll was removed, the leaf blades were stained in lugol‘s solution for the absence or presence of starch. All plants with lowered levels of GWD expression displayed a starch excess phenotype in leaves. Out of 30 independent transgenic plants screened only three lines (#16, #17 and #20) displayed a starch excess phenotype in leaves. In these lines the level of GWD expression was examined using western blot analysis. Seeds from all of these transgenic lines were sterilized and germinated on MS media (Murashing and Skoog, 1962) containing 50mg l-1 kanamycin. Seeds that were able to grow on this media were presumed to contain the transgene and positive transformants were planted in soil for further analysis.

3.10 Western Blot Analysis

Soluble proteins from the pericarp of 25 DAF old tomato fruits and leaves were denatured in buffer containing SDS Laemmli (1970). 25µg of soluble were separated by SDS-PAGE on either a 8% gel in the case of GWD protein or 10% gel in the case of FBPase and AGPase. The proteins were blotted at 4°C according to Khyse-Andersen (1984) on a nitrocellulose membrane (BA 85, 0.45µm; Schleicher und Schüll, Dassel) using a semi-dry electroblotting apparatus (MultiphorII, LKB, Bromma, Sweden). The blots were developed with rabbit serum


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followed by alkaline phosphatase-conjugated goat anti-rabbit serum (Amersham) according to Bhattacharyya et al. (1990).

3.11 RNA (Northern) Blot Analysis

Total RNA was isolated from frozen developing tomato fruits by a modification of the method of Hughes and Galau (1988). Plant tissue (5 to 10g) was ground to a fine powder in liquid nitrogen and sprinkled into 55 ml of ice-cold buffer (200mM Tris-HCL, pH 8.5, 300mM LiCl, 10mM EDTA, 1,5% (w/v) lithium dodecylsulfate, 1% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, 5% (w/v) insoluble PVP, 90mM â-mercaptoethanol, 10mM DTT, 0,5% (v/v) DEPC) and stirred to ensure immediate contact with the buffer. After stirring for 5 to 10 min, 46 ml of 3M ammonium acetate was added, and the extract was spun at 2500 g for 10 min at 4°C. RNA was precipitated from the supernatant with one-tenth volume of isopropanol and was centrifuged at 2500 g for 10 min at 4°C. The pellet was resuspended in 5 to 10 ml of H2O and purified by phenolchloroform extraction; this process was repeated until the preparation appeared clean. RNA was precipitated with ¼ volume of 10 M LiCl on ice for 2 to 12 hours, and then centrifuged at 2500 g for 10 min at 4°C. The RNA pellet was re-suspended in DEPC-water.

40 µg RNA was denatured in 40% (v/v) formamide, separated on a 1.5 % (w/v) agarose gel containing formaldehyde (Lehrach et al., 1977) and blotted onto nylon membrane (porablot NY plus, Macherey-Nagel, Düren, Germany), by means of capillary transfer using 20*SSC as the buffer (1*SSC is 0.15M NaCl, 0.015 M sodium citrate). The RNA was fixed to the membranes using an UV-crosslinker (Stratagene, La Jolla, USA). Hybridization of membranes was performed with 32P-labelled probes in 0.25M sodium phosphate (pH 7,2), 1mM EDTA, 1% (w/v) BSA and 7% (w/v) SDS. The filters were washed twice with 0.1 % SSC and 0.5% (w/v) SDS for 15 min at 68°C. The filters were subjected to autoradiography between intensifying screens at -80°C.

cDNA clones coding for plastidial transporters were either tomato EST‘s (TPT, EST No.cLEM23J19; ADP/ATP transporter, EST No.cLEM8I17) purchased from the Clemson University Genomics Institute (Clemson, South Carolina, USA) or in the case of Glc-6-P transporter, a potato clone which was the gift of Dr. Andreas Weber (Michigan State University, East lansing, Michigan, U.S.A). The plasmids were cut with sutable restriction enzymes and fragments isolated from a gel using the QIAquick kit (Qiagen) according to the manufacturers instructions. Radioactively labelled probes were made by the random primed


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method using a commercially available kit (Roche) according to the manufacturer instructions.

3.12 Determination of enzyme Maximum Catalytic Activities

3.12.1 Extraction Procedures and Assay Condition

Plant material was ground under liquid nitrogen to a fine powder in a pestle and mortar. Twice as much extraction buffer (50mM Hepes-KOH (pH 7.4), 5mM MgCl2, 1mM EDTA, 1mM EGTA, 10%(v/v) Glycerol, 0.1% (v/v) Triton X-100, 5mM DDT, 2mM epsilon-Amino-caproic acid, 2mM Benzamidine, and 0.5mM PMSF according to Trethewey et al. (1998), was added as weight of sample and the buffer and powder were mixed together. The samples were centrifuged at 2800 g and 4°C for 15 min and the supernatant was recovered. This was de-salted using NAP-5 columns (Pharmacia) and the resulting plant extract was either assayed for enzyme activity immediately in the case of AGPase and FBPase, or frozen in aliquots in liquid nitrogen before being stored at -80°C until use. Total protein content was determined by the method of Bradford (1976)

Extracts were kept at 4°C prior to assaying. If not noted otherwise, enzyme assays were carried out at 25°C in a final reaction volume of 300µl according to the accompanying references. The change in absorbance was continuously followed at 340nm using an Anthose ht II microtiter-plate reader (Anthos Labtec Instruments, Hanau).

Activities of Sucrose Phosphate Synthase and acidic Invertase were determined in stopped assays.

All coupling enzymes provided as ammonium sulfate suspension were desalted by centrifuging for 1 min, the supernatant being discarded and the sediment dissolved in the corresponding reaction buffer.

3.12.2 Phosphoglucoisomerase (EC 5.3.1.9)

Phosphoglucoisomerase was assayed in the direction of glucose-6-phosphate as described by Burrell et al., (1994). The assay consisted of 5µl de-salted extract in 75mM glycylglycine (pH 8.5), 10mM MgCl2, 0.5mM NAD+, 0.5U/ml glucose 6-phosphate dehydrogenase (Leuconostoc mesenteroides). The reaction was started by the addition of fructose 6-phosphate to a final concentration of 1mM.


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3.12.3 Phosphoglucomutase (EC 5.4.2.2)

Phosphoglucomutase was assayed in the direction of glucose-6-phosphate formation (Takamiya and Fukui, 1978). The assay consisted of 5µl de-salted extract in 50mM Hepes-KOH (pH 7.8), 5mM MgCl2, 2mM NAD+, 100µM glucose-1,6-bisphosphate, 0.5U/ml glucose 6-phosphate dehydrogenase (Leuconostoc mesenteroides). The reaction was started by the addition of glucose 1-phosphate to a final concentration of 2mM.

3.12.4 Hexokinase (EC 2.7.1.1)

Hexokinase was assayed in the direction of glucose-6-phosphate production as described by Veramendi et al., (1999). The assay consisted of 5µl de-salted extract in 50mM Tris/HCl (pH 8.0), 4mM MgCl2, 0.33mM NAD+, 2mM ATP, 2.5U/ml glucose 6-phosphate dehydrogenase (Leuconostoc mesenteroides). The reaction was started by the addition of glucose to a final concentration of 1mM.

3.12.5 Fructokinase (EC 2.7.1.4)

Fructokinase was assayed in the direction of glucose 6-phosphate as described by Renz et al., (1993). The assay contained 10µl de-salted extract in 50mM Tris/HCl (pH 8.0), 4mM MgCl2, 0.33mM NAD+, 2.5mM UTP, 2.5U/ml glucose 6-phosphate dehydrogenase (Leuconostoc mesenteroides) and 1.75 U/ml phosphoglucoisomerase (Yeast). The reaction was started by the addition of fructose to a final concentration of 1mM.

3.12.6 UDP-glucose Pyrophosphorylase (EC 2.7.7.9)

UDP-glucose pyrophosphorylase was assayed in the direction of glucose 1-phosphate formation (Zrenner et al., 1993). The reaction mixture contained 10µl de-salted extract in 100mM Tris/HCl (pH 8.0), 2mM MgCl2, 0.25mM NAD+, 2mM UDP-glucose, 20µM glucose-1,6-bisphosphate, 2.5U/ml glucose 6-phosphate dehydrogenase (Leuconostoc mesenteroides) and 3U/ml phosphoglucomutase (rabbit muscle). The reaction was started by the addition of tetrasodium pyrophosphate to a final concentration of 2mM.

3.12.7 Sucrose Synthase (EC 2.4.1.13)

Sucrose synthase was assayed in the direction of sucrose production (Sweetlove et al., 1996). The assay consisted of 5µl de-salted extract in 100mM Hepes-NaOH (pH 7.5), 4mM MgCl2, 0.2mM NADH, 40mM UDP-glucose, 1mM phosphoenolpyruvate, 10U/ml Pyruvate kinase


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and 2U/ml lactate dehydrogenase. The reaction was started by the addition of fructose to a final concentration of 10mM.

3.12.8 Enolase (EC 4.2.1.11)

Enolase was assayed in the direction of phosphoenolpyruvate production as described by Burrell et al., (1994). The assay consisted of 10µl de-salted extract in 100mM Hepes-NaOH (pH 7.5), 10mM MgCl2, 0.2mM NADH, 2.7mM ADP, 5U/ml Pyruvate kinase and 6U/ml lactate dehydrogenase. The reaction was started by the addition of 2-phosphoglycerate to a final concentration of 0.5mM.

3.12.9 Triose Phosphate Isomerase (EC 5.3.1.1)

Triose phosphate isomerase was assayed in the direction of dihydroacetone phosphate formation (Burrell et al., 1994). The assay consisted of 10µl de-salted extract in 100mM Hepes-NaOH (pH 8.0), 0.2mM NADH, 5mM EDTA, 1U/ml glycerol 3-phosphate dehydrogenase. The reaction was started by the addition of glyceraldehydes 3-phosphate to a final concentration of 1.5mM.

3.12.10 Phosphoglycerate Kinase (EC 2.7.2.3)

Phosphoglycerate kinase was assayed in the direction of formation 1,3-bisphosphoglycerate according to Burrell et al., (1994). The assay consisted of 5µl de-salted extract in 100mM Hepes-NaOH (pH 7.6), 2mM MgSO4, 0.3mM NADH, 1mM EDTA, 6.5mM glycerate 3-phosphate, 3.32U/ml glycerate 3-phosphate dehydrogenase. The reaction was started by the addition of ATP to a final concentration of 1mM.

3.12.11 Phosphofructokinase (EC 2.7.1.11)

Phosphofructokinase was assayed by the production of fructose-1,6-bisphosphate (Burrell et al.,1994). The assay consisted of 25µl de-salted extract in 100mM Tris/HCl (pH 8.0), 5mM MgCl2, 0.1mM NADH, 5mM fructose 6-phosphate, 1U/ml aldolase, 1.36U/ml glycerol 3-phosphate dehydrogenase and 2.6U/ml triose phosphate isomerase. The reaction was started by the addition of ATP to a final concentration of 1mM.

3.12.12 Pyruphosphate dependent Phosphofructokinase (EC 2.7.1.90)

Pyruphosphate dependent Phosphofructokinase activity was measured in the glycolytic direction by following the production of fructose-1,6-bisphosphate (Scott et al., 1995). The


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assay consisted of 10µl of de-salted extract in 75mM Hepes-NaOH (pH 7.5), 2mM Mg-acetate, 0.15mM NADH, 10µM fructose-2,6-bisphosphate, 7.5mM fructose 6-phosphate 1U/ml aldolase, 1.36U/ml glycerol 3-phosphate dehydrogenase (rabbit muscle) and 2.6U/ml triose phosphate isomerase (rabbit muscle). The reaction was started by the addition of tetrasodium pyrophosphate to a final concentration of 0.25mM.

3.12.13 Glyceraldehyde 3-Phosphate dehydrogenase (EC 1.2.1.12)

Glyceraldehyde-3-phosphate dehydrogenase was assayed in the direction of glyceraldehydes 3-phosphate production as described by Plaxton (1990). The assay consisted of 5µl de-salted extract in 100mM Hepes-NaOH (pH 8.0), 8mM MgSO4, 0.3mM NADH, 1mM EDTA, 2mM DTT, 6mM 3-phosphoglycerate and 4U/ml phosphoglycerate kinase. The reaction was started by the addition of ATP to a final concentration of 2mM.

3.12.14 Pyruvate Kinase (2.7.1.40)

Pyruvate kinase was assayed in the direction of pyruvate formation (Burrell et al., 1994). The assay consisted of 10µl de-salted extract in 50mM MOPS (pH 7.0), 15mM MgCl2, 0.15mM NADH, 100mM KCl, 5mM phosphoenolpyruvate and 6U/ml lactate dehydrogenase. The reaction was started by the addition of ADP to a final concentration of 5 mM.

3.12.15 Phosphoenolpyruvate Phosphatase (3.1.3.60)

Phosphoenolpyruvate phosphatase was assayed in the direction of pyruvate formation (Duff et al., 1989a). The assay used was the same as that for pyruvate kinase except ADP was omitted. The reaction was started by the addition of phosphoenolpyruvate to a final concentration of 5mM.

3.12.16 Fructose-1, 6-bisphosphatase (EC 3.1.3.11)

Fructose-1,6-bisphosphatase was assayed in the direction of fructose-6-phosphate production according to Kruger and Beevers (1984). The assay consisted of 40µl de-salted extract in 20mM Hepes-NaOH (pH 7.0), 5mM MgCl2, 0.5mM NAD+, 1U/ml phosphoglucoisomerase and 1U/ml glucose 6-phosphate dehydrogenase (Leuconostoc mesenteroides). The reaction was started by the addition of fructose-1,6-bisphosphate to a final concentration of 0.5mM.


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3.12.17 ADP-glucose Pyrophosphorylase (EC 2.2.7.27)

ADP-glucose pyrophosphorylase was assayed in the direction of glucose-1-phosphate formation as described by Müller-Röber et al., (1992). The assay consisted of 30µl de-salted extract in 80mM Hepes-NaOH (pH 7.4), 10mM MgCl2, 0.02%(w/v) fatty-acid free BSA, 0.6mM NAD+, 10µM glucose-1,6-bisphosphate, 10mM 3-phosphoglycerate, 3mM DTT, 1mM ADP-glucose 2.5U/ml phosphoglucomutase (rabbit muscle) and 1U/ml glucose 6-phosphate dehydrogenase (Leuconostoc mesenteroides). The reaction was started by the addition of tetrasodium pyrophosphate to a final concentration of 2mM.

3.12.18 Acid Invertase (EC 3.2.1.26)

Acid Invertase was assayed in the direction of sucrose degradation to produce glucose and fructose as described by Zrenner et al., (1996). The reaction mixture consisted of 20mM acetate buffer (pH 4.7), 100mM sucrose and 30µl of desalted enzyme extract in total volume of 100µl. The reaction mixture was incubated at 25°C for 60 min. After 1h, 25µl of 1M Tris/Hcl (pH 8.0) was added to the solution to be neutralized. The reaction mixture was stopped by heating at 95°C for 3 min. Control sample was prepared by heating the reaction mixture for 3 min in the presence of 25µl of 1M Tris/Hcl (pH 8.0) without period of incubation. For both control and assay samples glucose and fructose was measured directly in 25µl of the reaction mixture by the method of Stitt et al., (1989) as described below.

3.13 Determination of Soluble Sugars and Starch Content

Starch and soluble sugars glucose, fructose and sucrose were extracted as described by Trethewey et al., (1998) and determined photometrcally. The change in absorbance was continuously followed at 340nm using an Anthos hat II microtiter-plate reader (Anthos Labtec Instrument, Hanau).

Soluble sugars were determined modified from Stitt et al., (1989). The reaction mixture consisted of 5µl ethanolic extract and 250µl of 100mM imidazol, 5mM MgCl2, 2mM NADP+, 1mM ATP and 2U/ml glucose 6-phosphate dehydrogenase (yeast). To start the reaction, 5µl of the respective enzymes were sequentially added: for glucose 1U/ml hexokinase (yeast overproducer), for fructose 0.5U/ml phosphoglucoisomerase (yeast), for sucrose a 1:5 dilution of saturated solution of Invertase (beta-fructosidase from yeast).

Starch content was measured according to Trethewey et al., (1998) using a commercially available starch determination kit (Boehringer Mannheim, Mannheim). The assay is based on


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the enzymatic hydrolysis of starch by alpha-amyloglucosidase and the determination of glucose in a coupled assay with hexokinase and glucose 6-phosphate dehydrogenase.

3.14 Determination of Metabolic Intermediates

Trichloroacetic acid extracts of pericarp material for the determination of metabolic intermediates were prepared as described by Trethewey et al., (1998). The intermediates were determined photometrically in a final volume of 700µl according to Lytovchenko et al., (2002) using a Dual-wavelength spectrophotometer (ZWSII; Sigma, Berlin). Extraction procedure and assays were evaluated according to (Fernie et al., 2001).

Pyruvate and phosphoenolpyruvate were sequentially determined in 50mM Hepes-KOH (pH: 7.4), 5mM MgCl2, 50µM NADH and 1mM ATP. The reaction for pyruvate was started by addition of 0.6U lactate dehydrogenase (hog muscle), for phosphoenolpyruvate by addition of 2U pyruvate kinase (rabbit muscle).

Glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate, were determined in 50mM Hepes-KOH (pH: 7.4), 5mM MgCl2 and 250µM NADP+. The reactions were sequentially started by addition of 0.2U glucose 6-phosphate dehydrogenase (yeast), 0.4U phosphoglucomutase (rabbit muscle) and 0.4U phosphoglucoisomerase (yeast).

3-phosphoglycerate was assayed in 50mM Hepes-KOH (pH: 7.4), 5mM MgCl2, 50µM NADH, 1.5mM ATP and 5U/ml 3-phosphoglycerate kinase (yeast). The reaction was initiated by addition of 5U glyceraldehydes 3-phosphate dehydrogenase (rabbit muscle).

Inorganic phosphate was measured after extraction of the metabolite fraction in 700µl 3.5% perchloric acid as described by Sharkey and Vanderveer (1989). The extracts were neutralized to pH 6 to 7 by adding a solution of 2N KOH, 150mM Hepes (to help stabilize the pH), and 10mM KCl (to help the precipitation of KClO4). The phosphate assay was the malachite green enhanced-molybdate assay. An assay solution of 2g l-1 malachite green (Sigma M9636) and 10mM ammonium molybdate in 0.8 M HCl was made up at least two days prior to assay. This solution was filtered through Whatman No. 1 filter paper. Plant (10-50 µl) was added to 800µl of molybdate reagent. After 1 min, 100 µl 1M trisodium citrate was added to the assay. After 1 further min, 100µl of 1% Extran 1000 detergent was added to the assay. The optical density at 650 nm was read after 30 min and compared with standards made with dried KH2PO4.


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3.15 Analysis of fruit yield and flowers

3.15.1 Analysis of fruit weight

This trait was measured directly after harvesting when fruits were fully ripe (after 65 DAF). The weight of fruits was carried out on a balance.

3.15.2 Analysis of fruit size

This trait was also measured directly after harvesting when fruits were fully ripe (65 DAF). The size of fruits was carried out by a diameter.

3.15.3 Fruit setting

This trait was calculated by using the following formula:

Fruit setting% = No.of fruits that set/No.of flowers that anthesized *100

3.15.4 Date of 50% flowering

This trait was recorded as a number of days from date of planting to date of flowing 50% of plants.

3.16 Statistical Analysis of Data

t-tests were performed using the algorithm included into Microsoft Excel 2000. The expression ’significant‘ is used only when an alteration has been confirmed to be statistically significant ((P= 0,05) and (P= 0,01) with the Student‘s t-Test.


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