Cytokinin interconversion by StCKP1 controls potato tuber dormancy
Worldwide production of potatoes is in excess of 350 million metric tonnes per annum, of which 60% is intended for human consumption. Since the period of tuber dormancy before tuber buds sprout is usually shorter than the optimal market storage period, control of sprouting is essential. To prolong dormancy, tubers are either stored at low temperatures and/or are treated with chemical sprout inhibitors, the use of which subject to increasing scrutiny in the European Union due to their impact upon the environment. Cytokinins, a group of plant growth regulators, are known to play a central role in tuber bud sprouting and tuber initiation from stolon tips in Solanum tuberosum L. although it is unclear when and how cytokinins act to regulate dormancy. The interconversion of cytokinins is incompletely understood. Enzymes identified to date have higher affinities for aminopurines than their cytokinin equivalents. A novel cytokinin binding protein Solanum tuberosum Cytokinin Phosphorylase 1 (StCKP1), has been identified in tuberising stolon tips which shares regions of homology with members of the nucleosidase and phosphoribosyl transferase family. The composition of cytokinin N9 conjugates in tuber bud and surrounding tissue is known to change on transition from a dormant state, with an increase in base and riboside types observed. Analysis of transcripts indicates an increase in abundance of StCKP1 on tuberisation of stolon tips, and high abundance in the periderm of dormant tubers. Analysis of protein abundance by immunoblotting echoes this finding and indicates StCKP1 begins to accumulate in stolon tips shortly before tuberisation, matching binding activity. Transgenic analysis of the cytological reporter gene uidA under the control of two identified promoter regions indicates StCKP1 is expressed predominantly in tuber tissue. Analysis of StCKP1 activity by HPLC and LC-MS-MS shows that StCKP1 catalyses the interconversion of free base and riboside. KMs determined for cytokinin and aminopurine substrates indicate that StCKP1 has a higher affinity for cytokinin substrates and, of these cytokinins, displays a higher affinity for the free base catalysing ribosylation of the N9 to form the corresponding riboside. Desiree cultivars over-expressing StCKP1 under the CaMV 35S promoter exhibited an increased rate of tuberisation of stolon tips and an increase in the length of the dormant period following lifting. Over-expression of StCKP1 was found in particular to increase the chill sensitive period of dormancy, confirming results of StCKP1 knock-down by RNAi. Transcript abundance of StCKP1 at tuberisation in other cultivars including King Edward and Maris Peer was found to correlate with the dormancy characteristics prescribed by the European Cultivated Potato Database and the British potato variety database.