ULTRASTRUCTURAL LOCALIZATION OF OXIDASE ACTIVITIES IN CORN ROOT TIP CELLS WITH TWO NEW OSMIOPHILIC REAGENTS COMPARED TO DIAMINOBENZIDINE

The ultracytochemical potential of N,N'-bis(4-aminophenyl)-1,3-xylylene-diamine (BAXD) and N,N'-bis(4-aminophenyl)-N,N'-dimethylethylenediamine (BED) as electron donors for plant oxidases was tested and compared to 3,3'-diaminobenzidine (DAB). Incubations were 50-60 min, and osmication was for 60 min. Root meristematic cells which are rich in a variety of organelles and enzyme activities were used. Upon oxidation, BAXD produced intense staining of mitochondrial, amyloplast, Golgi and vacuolar membranes. Very intense homogenous staining of vesicles which varied in size and shape was also noted. BED in general was less readily enzymatically oxidized. This was seen as less intense or less frequent staining, particularly on mitochondrial cristae, when compared to BAXD. However, endoplasmic reticulum, plasmalemma and nuclear membrane were better stained with BED. Counterstaining was not used. In control experiments, inactivation by heat and by 2.5 hr of glutaraldehyde fixation were strong indications for the enzymatic nature of the oxidation. Experiments with catalase and peroxidase inhibitors provided evidence against the participation of these enzymes in the staining reactions. On the other hand, the oxidations were sensitive to cyanide (10 –3 M). This concentration prevented almost completely the oxidation of BAXD, BED and DAB. Azide (10 –2 M) was also an effective inhibitor, although less than 10 –3 M cyanide. Although the precise nature of BED oxidase is not yet known, it may be of interest to compare these results in plants with those already published for rat liver and rat heart (Seligman AM, Wasserkrug HL, Plapinger RE: Histochemie 23:63, 1970).