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DUO1 Is Required for AtCycB1;1 Expression in the Male
germline
The phenotype of duo1 shows that in addition to the activation of male germline
genes, DUO1 is required for germ cell division. Mutant duo1 germ cells com-
plete DNA synthesis (S) phase but fail to enter mitosis (M) [20],[24], suggesting
that DUO1 may regulate the expression of essential G2/M factors. As the Arabi-
dopsis CDK regulatory subunit AtCycB1;1 shows enhanced expression at G2/M
[25],[26] and is expressed in developing pollen, we investigated AtCycB1;1 as a
potential downstream target of DUO1. To monitor the expression of AtCycB1;1
we used the pCDG marker which contains the AtCycB1;1 promoter region and
mitotic destruction box fused to the
β
-glucuronidase (GUS) reporter [25]. First
we analysed the marker in wild type pollen (Figure 3A-F). Individual pollen
grains at different stages of development (as determined by DAPI staining) were
analysed for GUS activity, which results in the formation of indigo microcrystals.
Microspores and bicellular pollen shortly after mitosis contain numerous indigo
crystals, with the number peaking close to mitosis (Figure 3A-C), indicating that
expression of AtCycB1;1 is linked to asymmetric division. Expression is then abol-
ished in bicellular pollen (Figure 3D). Close to germ cell mitosis, single indigo
crystals are present specifically in germ cells (located by DAPI staining; Figure 3E)
indicating expression of AtCycB1;1 in the germ cell before division. The protein is
degraded after mitosis and is absent in tricellular pollen (Figure 3F).
We then counted the number of pollen grains with GUS staining at differ-
ent stages of development in wild type and heterozygous duo1 plants. In both
wild type and heterozygous duo1 plants, polarized microspores and vegetative
cells shortly after asymmetric division showed almost 100% staining, indicating
expression of AtCycB1:1 (Figure 3G). Thereafter vegetative cell staining declined
and was absent from late-bicellular stage pollen (Figure 3G). Germ cell staining
was subsequently observed in ~100% of pollen from wild type plants close to
mitosis, but was reduced by approximately half in heterozygous duo1 plants at
this stage (Figure 3H). As half of the pollen population is mutant in heterozygous
duo1 plants, and wild type pollen show GUS staining, this reduction in staining is
consistent with a lack of AtCycB1;1 expression in mutant duo1 pollen. This indi-
cates that DUO1 is required for the expression of AtCycB1;1 in male germ cells.
We then analysed the expression of AtCycB1;1 transcripts in seedlings after
steroid induction of mDUO1. In contrast to the germline markers, AtCycB1;1
was expressed at a low level in seedlings not exposed to estradiol and the presence
of DUO1 did not affect the level of AtCycB1;1 transcripts (Figure 2A). Thus,
although DUO1 is required for germline expression of AtCycB1;1 the presence of
DUO1 is not sufficient to induce AtCycB1;1 mRNA in seedlings. Transcription
of the AtCycB1;1 gene is known to be regulated by a number of factors, including
