Biology Reference
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female gametophytes yet apparently normal diploid ovule tissue, providing strong
support for the role of the haploid germ unit in promoting growth to the micro-
pyle [12,13]. Based on these studies, it was proposed that final stages of pollen
tube growth can be divided into two distinct phases: funicular guidance, in which
pollen tubes adhere to and grow up the funiculus, and micropylar guidance, where
pollen tubes enter the micropyle to deliver sperm to the female gametophyte [13].
Micropylar guidance signals originate at least in part from the two synergid cells
contained within the female gametophyte; pollen tubes do not enter ovules in
which synergid cells were either laser ablated [14] or defective due to a lesion in A.
thaliana MYB98 gene [15]. The maize EA1 protein, which is exclusively expressed
in the egg and synergids of unfertilized female gametophytes, may specify a role
for these cells in regulating micropylar guidance. Plants expressing EA1 RNAi or
antisense constructs produced significantly fewer seeds than wild type, and wild
type pollen tubes failed to enter mutant ovules [16].
In vitro assays have been used to characterize intracellular cues such as a Ca2+
gradient at the tip of pollen tubes that is critical for growth. Disrupting this gradi-
ent by iontophoretic microinjection or by incubation with Ca2+ channel blockers
can change the direction of tube growth [17]. The Ca2+ gradient in pollen tubes
is controlled by Rho GTPases; injection of antibodies against these proteins into
pollen tubes, or expression of dominant-negative forms of RhoGTPase, causes the
tip-focused Ca2+ gradient to diffuse and eliminates tube growth [18], presum-
ably by disrupting F-actin assembly [19]. These pollen tube growth defects can be
partially alleviated by adding high concentrations of extracellular Ca2+ [18].
In vitro grown pollen tubes also reorient their growth in response to certain
extracellular cues; lily pollen tubes are attracted to chemocyanin [4] and repelled
by a point source of nitric oxide [20]. In addition, in vitro grown pearl millet pol-
len tubes are attracted to ovary extracts [21]. For T. fournieri pollen tube guidance
across a simple medium and into the ovule was achieved after pollen tubes were
grown through a stigma and style [22]. In this species, the female gametophyte
protrudes from the ovule, and pollen tubes enter the micropyle without interact-
ing with funiculus [22]. Thus, the T. fournieri in vitro guidance system serves as
a model for the micropylar, but not the funicular guidance phase of pollen tube
growth to ovules [23]. Here, we describe an A. thaliana in vitro guidance assay
that recapitulates both funicular and micropylar guidance, serving as a model for
ovules with encased female gametophytes, an arrangement that is more common
among flowering plants. With the sequenced A. thaliana genome, the large col-
lection of mutants affecting reproductive functions, and comparative genomic
resources, this assay will greatly facilitate identifying genes that mediate the final
phases of pollen tube guidance.
