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After responding to the attractant and entering an ovule, the growth rate of
each pollen tube decreased to 0.98 ± 0.34 (s.d) µm/min (n = 19), reaching the
female gamtophyte and lysing after a 73 ± 19 (s.d) min delay (n = 19; Fig. 2g-l).
While previous work showed that pollen tube growth arrests only after reaching
the female gametophyte [9,10], our observations point to additional signals that
slow growth within the ovule prior to this arrest. This delay coincides with mean-
dering pollen tube navigation past the integument and nucellus cells (Fig. 2g-k),
potentially reflecting guidance by these cells.
Short-Range Guidance Signals from A. thaliana Ovules Are
Developmentally Regulated
The data presented above indicate that contact with A. thaliana stigmas and styles
enables pollen tubes to respond to diffusible ovule signals. To understand the na-
ture and source of these signals, we examined their activity during ovule develop-
ment. Previously it was shown that pollen tubes grow randomly or fail to elongate
in immature A. thaliana pistils [26]. However, it was impossible to distinguish
the contributions of distinct tissues in these experiments. Here, we exploited the
modular nature of the in vitro system, varying the age of stigmas, styles, and
ovules. While mature flower parts (stage 14 [27] were optimal, the stage of ovule
development was critical, with guidance factors completely absent at ~32 hrs
(stage 12a) and lower at ~16-24 hrs (stages 12b-c) before flowers mature (Table
2, upper panel). This pattern correlates with synergid development, the suggested
source of pollen tube attractants; these cells form after stage 12a [27,28]. Even so,
immature ovules promote better pollen tube guidance than heat-treated or fertil-
ized ovules, suggesting that a basal signalling capability is established early and
increases as the female gametophyte differentiates. In contrast, the developmental
stage of the stigma and style did not significantly alter targeting (
χ
2; P > 0.1;
Table 2, bottom panel), indicating that the signals that confer targeting com-
petence to pollen tubes do not vary over the course of pistil maturation (stages
12a-18) and that they emerge as early as stage 12a.
Short-Range Guidance Signals from Ovules Are Highly
Species-Specific
Like many traits that mediate reproduction [29,30], pollen tube guidance signals
diverge rapidly - crosses between A. thaliana and its relatives show random or ar-
rested pollen tube growth, even among species separated by <25 million years, MY
[13,31]. Because these interspecies crosses utilized intact pistils, it has been im-
possible to discern the roles of individual tissues; moreover, early blocks in pollen
