Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/19/2005
Publication Date: 11/1/2005
Citation: Suttle, J.C., Mornet, R. 2005. Mechanism-based irreversible inhibitors of cytokinin dehydrogenase. Journal of Plant Physiology. 162(11):1189-1196
Interpretive Summary: For an indeterminate period of time following harvest, potatoes will not sprout and are physiologically dormant. Dormancy is gradually lost during postharvest storage and the resultant sprouting is detrimental to the nutritional and processing qualities of potatoes. Because of this, sprouting results in severe financial loss to producers. In previous research from this lab, the plant hormone cytokinin was identified as the main dormancy breaking agent in potatoes. In order to identify how cytokinin content is controlled biochemically, specific inhibitors of cytokinin metabolism must be identified. In this paper, we show that a synthetic cytokinin analog irreversibly inhibited the activity of a key enzyme regulating cytokinin destruction. This compound effectively inhibited enzyme activity in the test tube and in plant tissues and can therefore be used to determine the role of this enzyme in controlling cytokinin levels during tuber dormancy. A more complete understanding of the roles of the naturally occurring plant hormones in tuber dormancy regulation and the internal mechanisms controlling their synthesis and action will greatly speed the processes of improving the activity of current sprout control agents and the identification of new sprout control agents/technologies.
Technical Abstract: The effects of three N6 -substituted aminopurine derivatives containing either allenic or acetylenic side-chains on in vitro and in vivo cytokinin deyhydrogenase (CKX: EC 18.104.22.168) activities were determined. At concentrations less than or equal to 100uM, the acetylenic derivative (HA-2) had no effect on in vitro CKX activity. In contrast, the two allenic derivatives (HA-1, and HA-8) inhibited in vitro CKX activity in a dose-dependent manner with 50% inhibition occuring at HA-1 and HA-8 concentrations of 9.0 and 0.4uM (respectively). HA-8 inhibited the degradation of both the free bases and ribosides on N6-(2-isopentenyl) adenine and zeatin. Pretreatment with HA-8 inhibited CKX activity in both a time- and concentration-dependent manner. In contrast to the reversible phenylurea inhibitor N-(chloro-4-pyridyl)-N-phenylurea, inhibition of CKX activity by HA-8 was not relieved by 24 hours of dialysis. Both HA-1 and HA-8 (but not HA-2) inhibited the metabolism of exogenous [3H]-N6-(2-isopentenyl) adenosine in excised aseptic potato (Solanum tuberosum L.) leaves. These results demonstrate that HA-8 is a mechanism-based irreversible (suicide) inhibitor of CKX and indicate that it may be useful in determining the role of CKX in cytokinin homeostasis in planta.