Genetic Subtraction Profiling Identifies Genes Essential for Arabidopsis Reproduction and Reveals Interaction Between the Female Gametophyte and the Maternal Sporophyte - Reproductive Physiology in Plants - page 89

and partial male sterility in the homozygous state (Vielle-Calzada J-P, Moore JM,
Grossniklaus U, unpublished data). Following tetrad formation three megaspores
degenerated, producing one viable megaspore, but megagametogenesis was not
initiated in coa. Despite the failure in embryo sac development, the integuments
and endothelium in coa differentiated similar to wild-type ovules (Figure 1). In
addition to our experiment with coa, we reanalyzed the dataset reported by Yu
and coworkers [34], who used the spl mutant and corresponding wild type for
a similar comparison. The spl mutant behaves both phenotypically and geneti-
cally very similar to coa [38]. The primary difference in the experimental set up
between the present study and that conducted by Yu and coworkers [34] is that
we did not dissect out the ovules from pistils, whereas Yu and coworkers extracted
ovule samples by manual dissection from the carpel, which led to a lower dilution
of 'contaminating' cells surrounding the embryo sac. However, our inclusion of
intact pistils allowed us to elucidate the carpel-specific and ovule-specific effects
controlled by the female gametophyte.
Figure 1. A genetic subtraction strategy for determination of the embryo sac transcriptome.
(a) A branch of a coatlique (coa) showing undeveloped siliques. Arrows point to a small silique, which bears
female sterile ovules inside the carpel (insert: wild-type Ler branch). (b) Morphology of a mature wild-type
ovule bearing an embryo sac (ES) before anthesis. (c) A functional embryo sac is absent in coa (degenerated
megaspores [DM]). Note that the ovule sporophyte is morphologically equivalent to that of the wild type. (d)
Functional categories of genes identified by a microarray-based comparison of coa and sporocyteless (spl; based
on data from Yu and coworkers [34]) with the wild type. The embryo sac expressed transcriptome is shown to
the left. Embryo sac expressed genes were grouped as preferentially expressed in the embryo sac if they were
not detected in previous sporophytic microarrays [28]. The size of the specific transcriptome in each class is
marked on each bar by a dark outline. Functional categories of genes that were identified as over-expressed in
the sporophyte of coa and spl are shown to the right. Scale bars: 1 cm in panel a (2 cm in the insert of panel a),
and 50 µ m in panels b and c.