Light microscopic localisation of aminoaldehyde dehydrogenase activity in plant tissues using nitroblue tetrazolium-based staining method

We have recently isolated pea aminoaldehyde dehydrogenase (AMADH, EC 1.2.1- ) and showed that it oxidises various co-aminoaldehydes, but not elementary aldehydes and betaine aldehyde. Now a simple method for specific staining in polyacrylamide gels was optimised enabling us to localise AMADH activity in plant tissues. When phenazine methosulphate (PMS) was used as a mediato r, AMADH in native PAGE gels readily reduced thiazolyl blue (MTT) or nitrob lue tetrazolium (NBT) producing the corresponding coloured formazans. Using NBT-based staining solution, AMADH activity was localised in cross section s of the root, hypocotyl, epicotyl and shoot apex of 7-d-old etiolated ea s eedlings. We followed a histochemical approach for the enzyme localisation in order to visualise tissues where the aminoaldehydes formed by the amine oxidase reaction are probably metabolised. In the root and hypocotyl, activ ity staining was most intense in cells belonging to the pericycle and endod ermis. Weaker staining (namely in the root) was observed in the vascular ca mbium. In both epicotyl and shoot apex, the major part of AMADH activity ap peared in vascular cambium cells. The violet formazan production was also o bserved in the pericycle and endodermis, but the staining intensity was low er. Pea amine oxidase, which produces naturally occurring aminoaldehydes as potential AMADH substrates, is known as an apoplastic enzyme associated wi th tissues undergoing lignification (xylem, sclerenchyma) and wall stiffeni ng (epidermis). Biological implications resulting from the localisation of both probably co-operating enzymes are discussed. (C) 2001 Editions scienti fiques et medicales Elsevier SAS.