Retargeting a maize β-glucosidase to the vacuole – Evidence from intact plants that zeatin-O-glucoside is stored in the vacuole

• Published in: Phytochemistry (Oxford) Vol. 79; pp. 67 - 77
• Main Authors: Kiran, Nagavalli S., Benková, Eva, Reková, Alena, Dubová, Jaroslava, Malbeck, Jiří, Palme, Klaus, Brzobohatý, Břetislav
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-07-2012
• Subjects: beta-Glucosidase - genetics; beta-Glucosidase - metabolism; Chloroplasts - drug effects; Chloroplasts - metabolism; Cytokinin metabolism; Cytokinins - metabolism; Glucosides - metabolism; Hybridization, Genetic; Nicotiana - cytology; Nicotiana - drug effects; Nicotiana - genetics; Nicotiana - metabolism; Nicotiana tabacum; Phaseolus - genetics; Plants, Genetically Modified; Seedlings - cytology; Seedlings - drug effects; Seedlings - growth & development; Storage organelle; Sub-cellular compartmentation; Tobacco; Vacuole; Vacuoles - drug effects; Vacuoles - genetics; Vacuoles - metabolism; Zea mays - enzymology; Zea mays - genetics; Zeatin - pharmacology; Zeatin-O-glucoside; β-Glucosidase; Cytokinin metabolism; Tobacco; β-Glucosidase; Zeatin-O-glucoside; Nicotiana tabacum; Vacuole; Sub-cellular compartmentation; Storage organelle
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2012.03.012

We have developed a molecular tool to probe cytokinin metabolism in planta using the robustness of the maize β-glucosidase Zm-p60.1 that cleaves zeatin-O-glucoside. [Display omitted]
► Plastid-localized Zm-p60.1 was successfully retargeted to the vacuole. ► Plants expressing Zm-p60.1 in the vacuole cannot store zeatin-O-glucoside. ► Vacuole-localized Zm-p60.1 restores zeatin-induced root-growth inhibition. ► The vacuole is the storage organelle for zeatin-O-glucoside. ► Zm-p60.1 is a robust probe for cytokinin metabolism in planta.
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 β-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.