Metabolic profiling to determine the cause of the increased triphenyltetrazolium chloride reduction in mannitol-treated maize callus

When Zea mays callus cultures of two different genotypes were treated with the osmoticum mannitol (0.53M) for 24h their ability to reduce the tetrazolium derivative 2,3,5-triphenyltetrazolium chloride (TTC) to form the insoluble red compound formazan is stimulated. The formazan can be extracted with 95% ethanol for quantitation and this reaction has been used as a measure of viability since only live cells can carry out this reduction. In order to determine the cause of the increased TTC reduction caused by mannitol we carried out metabolic profiling analysis using GC-MS to identify 80 compounds. There were increases in sugar alcohols, hexoses except fructose and in total sugars. The total organic acid pools did not change and nitrogen containing compounds decreased slightly. Principle component analysis showed a large treatment effect due to changes in carbohydrate and nitrogen metabolism. These results indicate that the increased carbohydrate available for the citric acid cycle may be the cause of the increased TTC reduction observed after the mannitol treatment.