Retargeting a maize ß-glucosidase to the vacuole--evidence from intact plants that zeatin-O-glucoside is stored in the vacuole.

Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize ß-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.

Involvement of hormones and KNOXI genes in early Arabidopsis seedling development.

Plant hormones control plant development by modulating the expression of regulatory genes, including homeobox-containing KNOXI genes. However, much remains to be elucidated about the interactions involved. Therefore, hormonal regulation of KNOXI gene expression was investigated using hormone applications and an inducible transgenic ipt expression system to increase endogenous cytokinin (CK) levels. Treatments with auxin, abscisic acid (ABA), cytokinins, ethylene, and gibberellin (GA) did not result in ectopic expression of the BP (BREVIPEDICELLUS) gene. However, BP expression was strongly reduced by ABA, increased by auxin treatment (correlating with the initiation of lateral root meristems, which strongly express BP), and did not significantly respond to short-term treatments with the other hormones in whole seedlings. Following short-term ipt activation, organ-specific differential regulation of KNOXI gene expression was observed. While several KNOXI genes were transiently up-regulated to low levels, STM was selectively repressed (especially at low light) in hypocotyls. In cotyledons, activation of CK-responsive genes preceded ipt induction, suggesting that CKs are transported more rapidly than the inducing agent (dexamethasone). Long-term increases in CK levels induced raised levels of several KNOXI transcripts in hypocotyls, correlating with the radial expansion of vascular tissues, the main domains of KNOXI gene expression, suggesting that CKs had little effect on KNOXI promoter activity. No alterations in hormone sensitivity were observed in a bp null mutant. Constitutive BP overexpression caused reductions in the length and number of lateral roots, while the primary root remained unaffected. The transgenic seedlings displayed weak, but significant, alterations in sensitivity to ABA, CK, and 1-amino-cyclopropane-1-carboxylic acid.