Phosphoglucose isomerase

To determine the levels of glucose, fructose, and sucrose, an enzymatic method was used as described previously [40 (link)]. An amount of 25 mg of lyophilized tomato fruits was mixed with 500 µL 80% (v/v) aqueous ethanol and incubated in a thermomixer at 1000 rpm at 80 °C for 1 h, followed by cooling on ice and centrifugation at 14,000× g at 4 °C for 5 min. The supernatant was collected and transferred to a new tube for evaporation to dryness (60–78 °C). The residue was dissolved in 250 µL water and subsequently used for sugar analysis in three technical replicates. Reactions were conducted in a 96-well plate with reaction mixtures each containing 100 µL diluted supernatant (7%) and 0.1 U glucose-6-phosphate dehydrogenase (Merck, Darmstadt, Germany) diluted in 100 µL buffer (100 mM Imidazole/HCl, pH 6.92, 10 mM MgCl₂, 2 mM ATP, 4 mM NAD). The reaction was performed in a microplate reader (Sunrise) and recorded at 340 nm wavelength for 3 min. Subsequently, 0.1 U hexokinase (Merck) was added to determine glucose level. The contents of fructose and sucrose were determined in the same way using 0.2 U phosphoglucose isomerase (Merck) and 1 U invertase (Merck), respectively. Levels were calculated using glucose, fructose, and sucrose as standards [23 (link)].

The determination of sugar levels was carried out according to Schaarschmidt [58 (link)] using 50 mg of root fresh weight. Reactions were done in triplicates using glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Merck, Darmstadt, Germany) in a microplate reader (Sunrise) and recorded at the wavelength of 340 nm. Then, hexokinase (Merck) was added to determine the level of glucose followed by addition of phosphoglucose isomerase (Merck) and invertase (Merck) to determine levels of fructose and sucrose, respectively. Values were calculated using glucose, fructose, and sucrose as standards.