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Cytokinins can be conjugated to N- and O-glucosides. While N-glucosylation generally leads to irretrievable loss of activity, O-glucosides are thought to play an important role in cytokinin storage and homeostasis since they can be hydrolyzed to the corresponding aglycones. The first zeatin O-glycosyltransferase genes isolated were ZOG1 and ZOX1 from beans (Martin et al., 1999a,b), and more recently cytokinin O- and N-glucosyltransferase genes were identified in Arabidopsis (Hou et al., 2005).
To study the effects of cytokinin O-glucosylation in monocots, maize (Zea mays) transformants harboring the ZOG1 gene under the control of the constitutive ubiquitin (Ubi) promoter were generated. The vegetative characteristics of hemizygous and homozygous Ubi:ZOG1 plants resembled those of cytokinin deficiency, including shorter stature, thinner stems, narrower leaves, smaller meristems, and increased root mass and branching. On the other hand, chlorophyll levels were higher and senescence was delayed in transformants when grown in the spring/summer. Unexpected modifications in reproductive development occurred as well. Hemizygous transformants had reduced tassels with fewer spikelets but normal viable pollen. Homozygotes had very small tassels and feminized tassel florets, resembling the tasselseed phenotype. This novel finding suggests a link between cytokinins and sex-specific floral development in monocots.
In order to better understand the dynamics of zeatin glucosylation, we studied the expression of the three major Arabidopsis glucosyltransferases, an O-glucosyltransferase (At1g22400) and two N-glucosyltransferases (At5g05860 and At5g05870) as well as three minor O-glucosyltransferases (At2g36750, At2g36790, and At2g36800) having very low specific activity with trans-zeatin in their native host. The spatial and temporal expression patterns in plants harboring promoter:GUS constructs showed some overlap, particularly in the root tips, but were also gene-specific. At1g22400 was consistently expressed in stomatal guard cells. RT-PCR indicated differential gene expression in response to stress and hormone treatments. At1g22400 was induced by drought stress supporting a possible role in stomatal control. Transformants harboring 35S:ORF:GFP constructs showed cytosolic and nuclear fluorescence, comparable to that found for 35S:GFP expression. Our findings demonstrate the functional significance of cytokinin glucosyltransferases by rapid regulation of active cytokinin levels in specific cell types. |
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