Plant Molecular Biology 39: 257–271, 1999.
© 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Characterization of the KNOX class homeobox genes Oskn2 and Oskn3
identiﬁed in a collection of cDNA libraries covering the early stages of rice
A. Dorien Postma-Haarsma, Ira I.G.S. Verwoert
, Oscar P. Stronk, Jan Koster, Gerda E. M.
Lamers, J. Harry C. Hoge and Annemarie H. Meijer
Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, P.O. Box 9505, 2300 RA Leiden,
author for correspondence);
Present address: Department of Genetics, Institute of Molecular
Biological Studies (IMBW), Vrije Universiteit, Biocenter Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam,
Received 17 March 1998; accepted in revised form 19 August 1998
Key words: developmental regulation, ectopic expression, embryogenesis, in situ hybridization, KNOX class
homeobox genes, rice
For identiﬁcation of genes involved in embryogenesis in the model cereal rice, we have constructed a collection
of cDNA libraries of well-deﬁned stages of embryo development before, during and after organ differentiation.
Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice
embryogenesis. Three types of KNOX clones were identiﬁed in libraries of early zygotic embryos. Two of these,
Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously
described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all
three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting
that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function
also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to
mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2
suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3
in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1
and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects
during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but
a more severly affected ﬂower morphology. The observation that the KNOX genes produce similar though distinct
phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target
genes, or that cell-type speciﬁc factors determine their precise action.
In the monocotyledonous Poaceae family containing
the agronomically important cereals, the process of
embryogenesis results in highly structured and rela-
The nucleotide sequence data reported will appear in the EMBL,
GenBank and DDBJ Nucleotide Sequence Databases under
the accession numbers AF050180 (Oskn2), AF050181 (Oskn3),
AF051153 (Osgb1) and AF51154 (Osgb2).
tively large mature embryos which have developed
several leaf primordia and a radicle, connected by a
vascular bundle system. Rice has become recognized
as a useful model system to study cereal embryoge-
nesis [8, 12, 14]. Based on studies of the available
rice embryo mutants, it was concluded that three ma-
jor regulatory processes, being positional regulation,
organ determination and size regulation, are opera-
tive in the very early stages of embryogenesis, prior