The pathway of betalain biosynthesis: Effect of cytokinin on enzymic oxidation and hydroxylation of tyrosine in Amaranthus tricolor seedlings

The effect of exogenously added tyrosine or l -3-(3,4-dihydroxyphenyl) alanine on the accumulation of betacyanin in response to cytokinin in Amaranthus tricolor (L.) var. tricolor half-seedlings depends on the age of the seedlings and the treatment of the seedlings prior to induction of pigment by benzyladenine. Neither extracted polyphenol oxidase, peroxidase or tyrosine hydroxylase activity, nor in vivo tyrosine hydroxylation is increased in response to exposure of seedlings to cytokinin. However a small percentage of the polyphenol oxidase activated or unmasked by Triton X-100 treatment of membrane fractions is increased after cytokinin treatment of half-seedlings for 22 h. It is concluded that cytokinin control may be on a multi-enzyme membrane-located complex involving part of the polyphenol oxidase activity of the tissue (possibly an isoenzyme), and that the majority of the polyphenol oxidase activity in Amaranthus is a constitutive membrane enzyme which is not involved in betacyanin synthesis. Although cytokinins do not affect in vivo tyrosine hydroxylation, this activity follows closely the accumulation of betacyanin which is first detectable about 6.5 h after the application of cytokinin. Only a very low level of in vivo hydroxylation can be demonstrated in half-seedlings treated for 6 h either with or without cytokinin but it begins to increase shortly after this time. A large increase in this activity by 16 h (independent of cytokinin) can be almost completely (79%) prevented by chloramphenicol (300 μM) although the drug increases accumulation of betacyanin. At this concentration about 30% of the protein synthesis in inhibited. In vitro tyrosine hydroxylation is, however, not reduced in half-seedlings treated with chloramphenicol during 16 h induction nor is extractable polyphenol oxidase reduced. It is concluded that chloramphenicol is inhibiting the synthesis of some protein essential for in vivo hydroxylation other than the activity measured during in vitro hydroxylation and that the inhibition of synthesis of 79% in vivo hydroxylation still leaves enough activity for maximum betacyanin synthesis.