Isolation of cDNAs coding for NtEPb1Á
b3, marker proteins for
pollen dedifferentiation in a tobacco pollen culture system
Masaharu Kyo *, Naoki Yamaji, Yasukazu Yuasa, Tsuyoshi Maeda, Hiroshi Fukui
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan
Received 26 March 2002; received in revised form 23 July 2002; accepted 29 July 2002
Several phosphoproteins, Nicotiana tabacum L. embryogenic pollen-abundant phosphoproteins (NtEPs), characteristically
appear in the dedifferentiation process from immature pollen grains to embryogenic cells in a pollen culture system. Among NtEPs
we focused our attention on three proteins (NtEPb1 Á
b3) which showed the highest correlation with the dedifferentiation and
possessed different pI values and similar molecular weights (ca. 22 kDa). Using probes designed from the N-terminal amino acid
sequence common to the three, we isolated 14 clones of cDNA belonging to three similar sequences which probably correspond to
b3. The predicted amino acid sequences showed moderate homology to NtEPc, several type-1 copper-binding
glycoproteins and a kind of early nodulin. The level of the transcripts for NtEPbs is highly associated with the pollen
dedifferentiation but not with pollen maturation nor with cell division accompanied by meiosis or proliferation of BY-2 cells. Such
an expression manner was distinguished from that of a gene coding for A-type cyclin (Ntcyc 25), indicating that NtEPb genes are
not under cell cycle control. These results suggest that there exist genes related to an unknown event other than the reentrance of cell
cycle in the dedifferentiation process of immature pollen that may be important for acquisition of embryogenic competence.
# 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Dedifferentiation; Nicotiana tabacum L.; Phosphoproteins; Pollen embryogenesis
Since the first observation in Datura innoxia ,
embryogenesis from pollen/microspore has been re-
ported in 240 species in 85 genera belonging to 38
families . Much effort has previously been done to
realize the efficient breeding system utilizing the andro-
genic dihaploid-population and also to provide insight
into the induction mechanism of the phenomenon .
The present situation of studies on the phenomenon is
summarized below according to recent reviews [4Á
It is well known that the productivity of androgenic
haploids in anther or pollen/microspore culture is
heritable and affected by genetic factors. In fact many
quantitative trait loci responsible for the productivity
have been identified in wheat, barley, corn, potato and
Brassica. However, no real probes or substantial
markers related to the loci have been obtained yet.
Many proteins associated with pollen/microspore em-
bryogenesis have been reported , but their roles in the
phenomenon have not been examined. Despite many
results obtained through various approaches in these
several years, our understanding of pollen/microspore
embryogenesis is still fragmented. To proceed the
understanding at a molecular level it seems to be
important to obtain more reliable markers for the
In some plant species, imposition of stress (for
example, malnutrition in tobacco, high temperature
treatment in rape, both combinations in wheat) acts as
a trigger for the induction of pollen/microspore embry-
ogenesis. This suggests that some stress-inducible genes
cause a change of the developmental program from
pollen maturation to embryogenesis. It seems to be
important to collect cDNAs associated with the phe-
The nucleotide sequences, seq. 12, 9 and 5 for NtEPb1 Á
respectively, reported in this paper have been submitted to DDBJ/
EMBL/GenBank under accession numbers AB080969-71.
* Corresponding author. Tel.: '
81-87-891-3132; fax: '
E-mail address: email@example.com (M. Kyo).
Plant Science 163 (2002) 1055 Á
0168-9452/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
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