Ectopic over-expression of the maize β-glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco

Abstract

The activity of the phytohormone cytokinin depends on a complex interplay of factors such as its metabolism, transport, stability, and cellular/tissue localization. O-glucosides of zeatin-type cytokinins are postulated to be storage and/or transport forms, and are readily deglucosylated. Transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants were constructed over-expressing Zm-p60.1, a maize beta-glucosidase capable of releasing active cytokinins from O- and N3-glucosides, to analyse its potential to perturb zeatin metabolism in planta. Zm-p60.1 in chloroplasts isolated from transgenic leaves has an apparent K(m) more than 10-fold lower than the purified enzyme in vitro. Adult transgenic plants grown in the absence of exogenous zeatin were morphologically indistinguishable from the wild type although differences in phytohormone levels were observed. When grown on medium containing zeatin, inhibition of root elongation was apparent in all seedlings 14 d after sowing (DAS). Between 14 and 21 DAS, the transgenic seedlings accumulated fresh weight leading later (28-32 DAS) to ectopic growths at the base of the hypocotyl. The development of ectopic structures correlated with the presence of the enzyme as demonstrated by histochemical staining. Cytokinin quantification showed that transgenic seedlings grown on medium containing zeatin accumulate active metabolites like zeatin riboside and zeatin riboside phosphate and this might lead to the observed changes. The presence of the enzyme around the base of the hypocotyl and later, in the ectopic structures themselves, suggests that the development of these structures is due to the perturbance in zeatin metabolism caused by the ectopic presence of Zm-p60.1.

Changes in cytokinin levels and metabolism in tobacco (Nicotiana tabacum L.) explants during in vitro shoot organogenesis induced by trans-zeatin and dihydrozeatin

“…Recently, Kiran et al (2006) described the possible hydrolysis of CK-O-glucosides by b-glucosidase, whereby the regulation of this enzyme activity might represent another mechanism involved in the functioning of bioactive CKs in plant organogenesis. The involvement of CKX in plant organogenesis has been previously reported by Auer et al (1999), who found an increase of CKX activity during the commitment to shoot induction in petunia leaf explants in the presence of BA.…”

“…T seeds were obtained from the T3 homozygous line of T tobacco. For a detailed description of Zm-p60.1 cloning, transformation, RT-PCR, kinetic analyses and associated properties see Kiran et al (2006). The tobacco seeds were surface-sterilized and sown in vitro on MS regulator-free medium (Murashige and Skoog 1962).…”

“…It has been suggested that explants derived from transgenic tobacco plants harboring the maize Zm-p60.1b-glucosidase gene (T) may have a higher organogenic capacity to that of wild-type (WT) plants, due to an easier release of bioactive CKs from O-glucosides as a result of the activity of over-expressed b-glucosidase gene product (Kiran et al 2006). Hence in the present study, the comparative analyses of the uptake and metabolism of two isoprenoid hydroxylated CKs (Z and DHZ) were conducted using T and WT tobacco plant leaf segments during the induction of shoot organogenesis.…”

The Biotechnological Potential of Cytokinin Status Manipulation

“…Tobacco plants that ectopically overexpress Zm-p60.1 had CK profiles that differed from those of wildtype plants but did not exhibit any morphological changes. However, when these mutants were treated with exogenous tZ, they accumulated higher levels of active CK metabolites than wild-type plants (Kiran et al 2006). This was accompanied by the inhibition of root elongation.…”

Focused directed evolution of β-glucosidases: theoretical versus real effectiveness of a minimal working setup and simple robust screening

“…7 Therefore, the present study focused on a well-characterized member of the b-glucosidases, the maize enzyme Zm-p60.1, as a model system. Zm-p60.1 (UNI-PROT ID: P49235; PDB ID: 1HXJ) plays a key role in the targeted release of active phytohormones (cytokinins) from their inactive storage forms (cytokinin-O-glucosides), [8][9][10] and thus in the regulation of plant development. Hence, there are profound reasons for acquiring information about its structure-activity relationships, in addition to the more generally relevant information that can be obtained from studying it.…”