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transformed with either of two control constructs (MGH3-AtCycB1;1::GFP,
which is not expressed in mutant pollen, and LAT52-AtCycB1;1, which is ex-
pressed only in the vegetative cell) did not vary significantly from 50% (Chi2
p<0.05) (Figure 3I). In contrast, in heterozygous duo1 plants transformed with
DUO1-AtCycB1:1 the majority of lines (31/49) showed a significantly reduced
frequency of bicellular pollen and a corresponding increase in tricellular pollen
(Figure 3I). This suggests that restoring AtCycB1;1 in duo1 mutant germ cells is
sufficient to promote mitosis in a proportion of the population. Complementa-
tion was however incompletely penetrant, which may result from the use of the
DUO1 promoter that may not produce native amounts of AtCycB1;1. It is also
possible that other factors with a role in G2/M transition, such as other AtCycB
family members that are also expressed during pollen development [31], may also
be absent in duo1 pollen.
To determine if the presence of DUO1-AtCycB1;1 in duo1 pollen restored
only the ability to proceed through mitosis or germline specification as well, we
analysed expression of the AtMGH3 and AtGCS1 markers in duo1 plants show-
ing partial complementation (Figure 3J). In contrast to plants without DUO1-
AtCycB1;1 where almost all tricellular pollen expresses GFP, plants displaying
partial complementation produce ~10% of pollen that is tricellular but does not
express the markers. As there is also a ~10% decrease in bicellular pollen, this new
class of tricellular pollen is most likely duo1 pollen in which the division defect
has been complemented by the DUO1-AtCycB1;1 construct, but in which the
markers have not been activated. Consistent with this, DUO1-AtCycB1;1 com-
plemented duo1 pollen showed no male transmission. Thus, complementation
of the bicellular phenotype by AtCycB1;1 only affects cell division and does not
restore expression of germline gene expression and sperm cell function.
DUO1 Expression Is Restricted to the Male Germline
Independent of a Putative GRSF Binding Site
Closer examination of mature pollen grains ectopically expressing DUO1 in the
vegetative cell revealed a distinctive morphology with reduced cytoplasmic den-
sity, larger vacuoles and numerous large cytoplasmic inclusions. This phenotype
was only found in pollen containing vegetative nucleus GFP and analysis of pol-
len viability revealed up to 50% non-viable pollen with the aberrant pollen not
being viable. Similar phenotypes are not seen in pollen of plants transformed with
LAT52-H2B::GFP where the transgene is transmitted normally (data not shown).
Furthermore, Arabidopsis plants constitutively expressing DUO1 (driven by the
35S promoter) show severe seedling patterning defects, twisted and curled leaves
and floral defects [21]. These phenotypes demonstrate the importance of restrict-
ing high level expression of DUO1 to male germ cells.
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