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Such restriction may partially rely upon degradation of DUO1 mRNA by
microRNA159 [21] in certain cell types but promoter elements are also likely to
be important. As such, restriction of DUO1 expression to the male germline has
been proposed to rely on the repressor protein GRSF due to a putative GRSF
binding site in the DUO1 promoter [14]. Mutagenesis of similar sequences in
the LGC1 promoter led to ectopic activation of the LGC1 promoter in non-germ
line cells in tobacco and Arabidopsis [14]. However, when we specifically mutated
the putative GRSF binding site in the DUO1 promoter this did not affect the ger-
mline-specific expression of DUO1 (Figure 4A-D). Moreover, sequences in the
150 bp proximal DUO1 promoter, excluding putative GRSF binding sites, were
sufficient for germline-specific expression (Figure 4E). Although factors that bind
to the lily LGC1 silencer appear to be present in non-germline cells in Arabidop-
sis [14], the germline-restricted activation of DUO1 does not appear to involve
GRSF mediated repression. Since the DUO1 promoter appears to be active only
after asymmetric division in the newly formed germ cell and that activation does
not depend upon DUO1 itself (see Figure 1), activation of the DUO1 promoter
may depend on proximal region-binding transcription factors that are inherited
and/or segregated during asymmetric division of the microspore.
Figure 4. Male germline specificity of DUO1 does not depend on putative GRSF binding sites.
(A) Schematic of the DUO1 promoter region illustrating the mutagenized putative GRSF binding site. (B,C)
Expression of H2B::GFP in pollen driven by the native (B) or mutagenized DUO1 (C) promoters. Top
panels show GFP signal, lower panels show DAPI staining. (D) RT-PCR analysis of native and mutagenized
DUO1 promoter activity in seedlings. PCR was conducted on cDNA from wild type plants (1), control plants
transformed with a constituitive HistoneH3 promoter-H2B::GFP fusion (2), and plants transformed with the
native (3), or mutagenized (4), DUO1 promoters driving H2B::GFP expression. The primers used were specific
for GFP (upper panel) or native Histone H3 transcripts (lower panel). The native or mutagenized DUO1
promoters showed no sporophytic expression of GFP transcripts. (E) Schematic representation of the of the
DUO1 promoter 5 deletion series used to drive expression of H2B::GFP. The first four deletions, including
deletion 3 in which the putative GRSF binding site is removed, showed a similar expression pattern to that of the
full-length DUO1 promoter, with GFP signal only observed in sperm cell nuclei. The same expression pattern
was observed in all independent lines examined (n). GFP expression was not observed in any transformants
harbouring the shortest promoter fragment (deletion 5).
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