Plant Molecular Biology 51: 237–248, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
237
Functional characterisation of a cytokinin oxidase gene DSCKX1 in
Dendrobium orchid
Shu Hua Yang, Hao Yu and Chong Jin Goh
∗
Plant Growth and Development Laboratory, Department of Biological Sciences, Faculty of Science, National
University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Republic of Singapore (
∗
author for
correspondence; e-mail: dbsgohcj@nus.edu.sg)
Received 16 December 2001; accepted in final form 24 May 2002
Key words: cytokinin, cytokinin oxidase, Dendrobium orchid, DSCKX1, shoot/root development
Abstract
Cytokinin oxidase plays an important role in the cytokinin regulatory processes. We have cloned a novel putative
cytokinin oxidase, DSCKX1 (D
endrobium Sonia cytokinin oxidase), by mRNA differential display from shoot
apices of Dendrobium Sonia cultured in the presence of BA. The DSCKX1 gene appears to have three alternative
splicing forms and its expression of DSCKX1 was induced in a tissue-specific manner by cytokinins. In transgenic
orchid plants overexpressing DSCKX1, the elevated level of cytokinin oxidase activity was accompanied by a
reduction of cytokinin content. These plants exhibited slow shoot growth with numerous and long roots in vitro.
Their calli also showed decreased capability of shoot formation. Conversly, antisense transgenic plants showed
rapid proliferation of shoots and inhibition of root growth combined with a higher endogenous cytokinin content
than wild-type plants. Thus DSCKX1 appears to play an important role on cytokinin metabolism and the related
developmental programmes in orchid.
Abbreviations: BA – benzylaminopurine; CaMV – cauliflower mosaic virus; CKX – cytokinin oxidase; DIG –
digoxigenin; FAD – flavin adenine dinucleotide; iP – isopentenyladenine; iPA – isopentenyl adenosine; ZR –
zeatin riboside.
Introduction
Cytokinins are essential phytohormones involved in
many processes of plant growth and development,
including cell division, shoot development, growth
of lateral buds, photosynthesis, chloroplast devel-
opment, and delay of leaf senescence (Skoog and
Armstrong, 1970; Mok, 1994). Therefore, it is es-
sential to understand the biosynthetic and metabolic
regulation of cytokinins in plant cells for a better
comprehension of regulatory circuits involved in the
equilibrium of hormone levels and the corresponding
effect on plant growth and development. Although
the molecular mechanisms involved in the control of
cytokinin homeostasis are far from being fully eluci-
dated, much recent progress has opened up possibil-
ities for addressing this problem. Several genes en-
coding adenylate isopentenyltransferase, a cytokinin
biosynthesis enzyme, have been recently identified
and characterised in Arabidopsis (Kakimoto, 2001;
Takei et al., 2001). Some of the genes encoding
purine metabolism enzymes, which act in the inter-
conversion of cytokinins, have been isolated from
Arabidopsis and Physcomitrella patens (Moffatt et al.,
1992; Schnorr et al., 1996; von Schwartzenberg
et al., 1998). There are several identified processes
involved in metabolism of cytokinins, which mainly
include N-glucosylation and O-glucosylation by spe-
cific glucosyltransferases, and N
6
-side-chain cleavage
by cytokinin oxidase (CKX) (Letham and Palni, 1983;
Brzobohatý et al., 1994). Three genes, encoding
enzymes O-xylosyltransferase, O-glucosyltransferase,
and cis-zeatin O-glucosyltransferase, were cloned
from Phaseolus vulgaris, P. lunatus,andZea mays,
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