Submitted to: Journal of Experimental Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2009
Publication Date: 5/12/2009
Citation: Smehilova, M., Galuszka, P., Bilyeu, K.D., Jaworek, P., Kowalska, M., Sebela, M., Sedlarova, M., English, J.T., Frebort, I. 2009. Subcellular Localization and Biochemical Comparison of Cytosolic and Secreted Cytokinin Dehydrogenase Enzymes from Maize. Journal of Experimental Botany. 60(9):2701-2712. Interpretive Summary: Plant hormones control many developmental processes in the plant. The cytokinin class of plant hormones have been demonstrated to have roles in cell division and other growth processes. The levels of cytokinins present in any specific location of the plant is dependent on the rate of synthesis, transport, uptake, and metabolism. The enzymes responsible for cytokinin metabolism are cytokinin dehydrogenases, and they irreversibly degrade the hormone to inactive components. All plant genomes investigated to date appear to contain multiple versions of cytokinin dehydrogenases that are secreted into the apoplast cell compartment. In contrast, plant genomes appear to only contain one version of cytokinin dehydrogenase enzyme that does not have a signal sequence to direct it to a subcellular compartment. In this manuscript, the maize version of the non-secreted cytokinin dehydrogenase was characterized. The objective of this work was to develop a better understanding of the localization of the maize enzyme and to characterize and compare the biochemical properties with a maize secreted cytokinin dehydrogenase. The non-secreted enzyme was localized in the plant cytosol, and it had different biochemical properties than the secreted version. The impact of this work is to develop a target for altering plant growth and development through manipulation of a single gene that may have a non-redundant function in plants.
Technical Abstract: Cytokinin dehydrogenase (CKX, EC 126.96.36.199) degrades cytokinin hormones in plants. There are several differently targeted isoforms of CKX in cells of each plant. While most CKX enzymes appear to be localized in the apoplast or vacuoles, there is generally only one CKX per plant genome that lacks a translocation signal and presumably functions in the cytosol. The only extensively characterized maize CKX is the apoplastic ZmCKX1; a maize gene encoding a non-secreted CKX has not previously been cloned or characterized. Thus, the aim of this work was to characterize the maize non-secreted CKX gene (ZmCKX10), elucidate the ZmCKX10 subcellular localization, and compare its biochemical properties with ZmCKX1. An expression profile of ZmCKX1 and ZmCKX10 was performed in maize tissues to determine the transcript abundance and organ-specific expression. For determination of subcellular localization, the CKX genes were fused with green fluorescent protein (GFP) and overexpressed in tomato hairy roots. Using confocal microscopy, ZmCKX1-GFP signal was confirmed to be present in the apoplast, whereas ZmCKX10-GFP was detected in the cytosol. No interactions of ZmCKX1 with the plasma membrane were observed. While roots overexpressing ZmCKX1-GFP formed significantly more mass in comparison to the control, non-secreted CKX overexpression resulted in a small reduction in root mass accumulation. Biochemical characterization of ZmCKX10 was performed using recombinant protein produced in Pichia pastoris. In contrast to the preference for DCPIP as an electron acceptor and trans-zeatin, iP and iPR as substrates for ZmCKX1, the non-secreted ZmCKX10 had a range of suitable electron acceptors, and the enzyme had a higher preference for cis-zeatin and cytokinin N-glucosides as substrates.