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mRNA was not detectable in floral tissues both using in situ hybridization and
RT-PCR (Figure 3N, Figure 4A-B). No signal was detected in floral tissues with
sense probes of OpdCYC1C (Figure 3O) and OpdCYC2A (Figure 3P).
To further elucidate the role of OpdCYC genes in floral development of
Opithandra dinghushanensis, we carried out semiquantitative RT-PCR studies
of two OpdcyclinD3 genes because cyclinD3 genes were previously revealed to
be negatively controlled by CYC as shown in the mid-to-late stage flowers in the
model organism snapdragon [6]. RT-PCR results showed that OpdcyclinD3a was
strongly expressed in lateral stamens from middle to late stages while its weak
mRNA signal was also detected in lateral and ventral petals (Figure 4A-B, E).
OpdcyclinD3a mRNA was not detected either in the dorsal region (the dorsal
petals and staminode) and ventral staminodes (Figure 4A-B, E). OpdcyclinD3b
transcripts were widely distributed in floral tissues except ventral staminodes, in
which its mRNA signal was strong in lateral stamens from middle to late stages
(Figure 4A-B, F). Transcripts of OpdcyclinD3b detected in the dorsal region were
likely mainly distributed in the dorsal petals (OpdcyclinD3b mRNA was uneasily
detectable in the dorsal staminode using in situ hybridization (data not shown)).
Discussion
The androecium of Opithandra only has two fertile stamens at the lateral positions
with three sterile stamens (staminodes) at the dorsal and ventral sides (Figure 1)
(also see [24]). The dorsal aborted stamen is tiny and barely detectable at anthesis
while the two infertile ventral stamens have short filaments with very small and
sterile anthers (Figure 1B-C). Correlative with the differentiation along the dors-
oventral axis of the morphologically peculiar androecium, the OpdCYC2A strong
expression is restricted to the dorsal staminode while OpdCYC1C transcripts are
initially distributed in all five stamen primordia but later are concentrated in the
dorsal and ventral staminodes to late stages. In the ECE lineage of CYC/TB1
subfamily, the TCP proteins in ECE-CYC2 clade studied to date function as
negative regulators in stamen development, whereas they appear to vary in petal
development according to the trait concerned [3-5,9,10,12,17,23,25,34,35]. The
abortion of the dorsal stamen in Antirrhinum comes from CYC and DICH ac-
tivities [4,5]. The CYC-like gene expression expansion from the dorsal to both the
dorsal and lateral stamens is correlated with abortion of both the dorsal and lat-
eral stamens in Mohavea and Chirita [23,25]. TB1 gene exhibits a mix feature of
ECE-CYC1 and ECE-CYC2 clades and functions to suppress axillary meristem
(CYC1) while retard stamen growth (CYC2) in maize [3,12,34]. In Asteraceae, a
CYC homologue GhCYC2 from Gerbera functions by disrupting stamen devel-
opment [17]. Given that CYC-like gene (ECE-CYC2 clade) function is conserved in
