Biological activity of cytokinins derived from Ortho- and Meta-Hydroxybenzyladenine

To determine the structure-activity relationships of natural aromatic cytok inins, the activity of 6-benzylaminopurine (BAP) and its hydroxylated deriv atives was compared in three bioassays based on stimulation of tobacco call us growth, retention of chlorophyll in excised wheat leaves, and dark induc tion of betacyanin synthesis in Amaranthus cotyledons. The aromatic cytokin ins 6-(2-hydroxybenzylamino)purine (ortho-topolin) and 6-(3-hydroxybenzylam ino)purine (meta-topolin), their 9-ribosides and 9-glucosides, were synthes ized by the condensation of 6-chloropurine and its 9-glycosides with the ap propriate hydroxybenzylamine. The activity of free bases, 9-ribosides and 9 -glucosides was compared with that of BAP, trans-zeatin and their 9-glycosi des. Hydroxylation of the benzyl ring in the meta position increased the ac tivity of BAP and its riboside in tobacco callus and chlorophyll retention bioassays, whereas ortho-hydroxylation decreased the activity. In contrast, in the Amaranthus bioassay meta-hydroxylation of BAP substantially decreas ed its activity. Ribosylation at position 9 had no significant effect on th e activity of zeatin, BAP and both topolins. The activity of 9-glucosides o f all cytokinins tested was near zero. The biological activity of meta-topo lin and its riboside is comparable to that of the most active isoprenoid cy tokinin, zeatin, in tobacco callus growth and senescence bioassays. The res ults establish the existence of a family of endogenous aromatic cytokinins centered around the highly active compound, meta-topolin. We also report he re an improved chlorophyll retention bioassay based on incubation of 2.5 cm long detached wheat leaf segments in microtiter plate wells containing 150 mu l of cytokinin solution. The consumption of cytokinin to be tested is 0 .1 mu mol per assay only. The amount as small as 1.5 pmol of substance can be estimated using this biotest.