Plant Molecular Biology 53: 227–236, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
Cyclin D-knockout uncouples developmental progression from sugar
Stefan Lorenz, Stefanie Tintelnot, Ralf Reski and Eva L. Decker
Freiburg University, Plant Biotechnology, Schaenzlestrasse 1, 79104 Freiburg, Germany (
author for correspond-
ence; e-mail firstname.lastname@example.org)
Received 14 May 2003; accepted in revised form 23 September 2003
Key words: cell cycle, CycD, development, knockout, Physcomitrella
Multicellular organisms need to modulate proliferation and differentiation in response to external conditions. An
important role in these processes plays the mitogen-stimulated induction of cyclin D(cycD) gene expression. D-
type cyclins have been identiﬁed as the crucial intracellular sensors for cell-cycle regulation in all eukaryotes.
However, cycD deletions have been found to cause speciﬁc phenotypic alterations in animals but not yet in plants.
An insertional mutation of a so far uncharacterized Arabidopsis cycD gene did not alter the plant phenotype. To
gain new insights into CycD function of land plants, we generated targeted cycD gene knockouts in the moss
Physcomitrella patens and observed a surprisingly limited disruption phenotype. While wild-type plants reacted to
exogenous glucose sources with prolonged growth of juvenile stages and retarded differentiation, cycD knockouts
exhibited developmental progression independent of sugar supply. On the other hand, growth rate, cell sizes or
plant size were not affected. Thus, we conclude that Physcomitrella CycD might not be essential for cell-cycle
regulation but is important for coupling the developmental progression to nutrient availability.
Abbreviations: CDK, cyclin-dependent kinase; CTAB, cetyltrimethylammonium bromide; CycD, cyclin D; EDTA,
ethylenediaminetetraacetic acid; EST, expressed sequence tag; G418, Geneticin; PEG, polyethylene glycol; SDS,
sodium dodecyl sulfate; Rb, retinoblastoma protein
A highly coordinated progression through the cell
cycle in response to environmental conditions is of
crucial importance for growth and development of all
multicellular organisms. For plants, the inﬂuence of
certain cell-cycle regulators on differentiation events
has been described recently (Riou-Khamlichi et al.,
1999; Rossi and Varotto, 2002).
One of the key regulatory points within the euk-
aryotic cell cycle displays the G1/S transition, which
is controlled by the highly conserved retinoblastoma
pathway in animals as well as in higher plants (Sherr,
1993; Francis, 1998; Burssens et al., 1998). Here,
a binary complex of a D-type cyclin (CycD) and a
cyclin-dependent kinase (CDK-a) provides the com-
mitment to the G1/S transition by phosphorylation
of the retinoblastoma protein (Rb). Rb phosphoryla-
tion subsequently causes the release of active E2F
transcription factors, which are required for S-phase
progression (den Boer and Murray, 2000).
Within this pathway the D-type cyclins are be-
lieved to provide the complex interconnection between
sensing of environmental signals and cell-cycle de-
cisions of cellular growth and differentiation, respect-
ively (Meijer and Murray, 2000; Oakenfull et al.,
2002). Expression of cycD genes is stimulated by mi-
togens, i.e. growth factors in mammals (Matsushime
et al., 1991; Sherr, 1994) and phytohormones in plants
(Riou-Khamlichi et al., 1999; Gaudin et al., 2000;
Hu et al., 2000; Oakenfull et al., 2002). In addition,
sucrose has been shown to differentially regulate D-
type cyclin expression in Arabidopsis regarding both
kinetics and the rate of induction (Riou-Khamlichi