Biology Reference
In-Depth Information
Results/Discussion
DUO1 Is a Key Regulator of Sperm Cell Specification
To investigate the potential role of DUO1 in regulating sperm specification we
examined the expression of three male germline markers, AtMGH3, AtGEX2
and AtGCS1, in mutant duo1 pollen. We exploited marker lines with promoter
regions of these germline genes linked to GFP. First we characterised the expres-
sion of these markers in a coordinated manner using confocal laser scanning mi-
croscopy (CLSM) throughout development of wild-type pollen (Figure 1A-C),
and compared their profiles with the expression of a DUO1:mRFP fusion protein
under control of the DUO1 promoter (DUO1-DUO1::mRFP; Figure 1D). The
expression of all three germ cell markers is undetectable in free microspores when
DUO1 is not expressed (Figure 1, Panel 1). Fluorescence is first detected in the
germ cell during or soon after engulfment by the vegetative cell, appearing at a
similar time to the expression of DUO1 (Figure 1, Panel 2). As the pollen ma-
tures the level of GFP accumulates in germ cells before mitosis and remains high
in mature sperm cells (Figure 1A-C, Panels 3-5). The accumulation of GFP in
progressive stages is illustrated by the reduced autofluorescence signal arising from
the pollen wall, reflecting the reduced exposure needed to capture a relatively
unsaturated germ cell GFP signal. DUO1 expression persists during pollen de-
velopment, although its abundance does not obviously increase in tricellular and
mature pollen (Figure 1D). Our analysis shows that in common with AtMGH3
and AtGEX2, the expression of AtGCS1, previously thought to be sperm cell-spe-
cific in Arabidopsis [11], is detected in germ cells soon after asymmetric division
(Figure 1C). The expression of all male germ cell markers shortly after the asym-
metric division shows that sperm cell specification begins early after inception of
the germline prior to passage of germ cells through mitosis.
The three male germline markers were introduced into heterozygous duo1
plants that produce 50% wild type pollen and 50% mutant pollen, and GFP
expression was scored. Virtually all the wild type pollen showed GFP fluorescence
in twin sperm cells while there was no fluorescence, or rarely a weak GFP signal,
in the single germ cell in duo1 pollen (Figure 1E-G, I-K). When these markers
were introduced into the cdka;1 mutant in which the arrested germ cell is able
to fertilize the egg cell, fluorescence was observed in the single germ cells in mu-
tant pollen (Figure 1E-G). This result confirms that germ cell division and cell
fate specification are uncoupled in cdka;1 mutant pollen, similar to the observed
expression of germ cell markers in arrested but functional germ cells in CAF1
mutants [19]. The absence of GFP in mutant duo1 germ cells demonstrates that
DUO1 is necessary for the expression of several germline-expressed genes, and
explains why duo1 pollen is infertile (it lacks proteins including AtGCS1 that
