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Journal of Experimental Botany, Vol. 53, No. 367, pp. 361-370, February 1, 2002
© 2002 Oxford University Press

In situ staining of activities of enzymes involved in carbohydrate metabolism in plant tissues

Lidiya I. Sergeeva1,2 and Dick Vreugdenhil1,3

1 Laboratory of Plant Physiology, Graduate School of Experimental Plant Sciences, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
2 Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya 35, 127 276, Moscow, Russia

A powerful technique is described to localize the activities of a range of enzymes in a wide variety of plant tissues. The method is based on the coupling of the enzymatic reaction to the reduction of NAD and subsequent reduction and precipitation of nitroblue tetrazolium. Enzymes that did not reduce NAD could be visualized by coupling their activities to glucose-6-phosphate dehydrogenase activity via one or more intermediary ‘coupling’ enzymes. The method is shown to be applicable for the detection of the activities of hexokinase, fructokinase, sucrose synthase, uridine 5'-diphospho-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase, phosphoglucomutase, and phosphoglucose isomerase. It could be used for all tissues tested, including green leaves, stems, roots, fruits, and seeds. The method is specific, very sensitive, and has a high spatial resolution, giving information at the cellular and the subcellular level. The localization of sucrose synthase, invertase, and uridine 5'-diphospho-glucose pyrophosphorylase in transgenic potato plants, carrying a cytokinin biosynthesis gene, is studied and compared with wild-type plants.

Key words: Carbohydrate metabolism, enzymes, in situ staining, plant tissues.


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