IDENTIFICATION OF GENES EXPRESSED IN THE

FEMALE GAMETOPHYTE

Currently, very few genes expressed in the female gametophyte

have been isolated (Springer et al., 1995; Kranz and

Dresselhaus, 1996; Nadeau et al., 1996; Perry et al., 1996).

However, several of the female gametophyte mutations

 were created by T-DNA (e.g., the fem and

gfa mutations) or transposon (e.g., the hdd mutation) inserts.

This will facilitate the rapid cloning of the corresponding

genes, which appear to control specific steps of megagametogenesis

including cellularization and the fusion of polar

nuclei.

Identifying genes expressed in specific cells of the embryo

sac has been hampered by difficulties in isolating female gametophytes,

which are small and relatively inaccessible.

These limitations have also frustrated molecular and biochemical

experiments. However, new approaches in maize

and Arabidopsis are beginning to overcome these problems.

For example, in maize, procedures for isolating large numbers

of viable female gametophytes and egg cells have been

developed (Wagner et al., 1988; Kranz et al., 1991). This advance

has allowed the construction of cDNA libraries from

unfertilized egg cells (Dresselhaus et al., 1994) and the isolation

of several clones that appear to be expressed exclusively

in the egg cell (Kranz and Dresselhaus, 1996). These

procedures could also be applied to isolate genes expressed

specifically in the other female gametophyte cells

(Dumas and Mogensen, 1993).

In Arabidopsis, a large collection of gene-trap and enhancer-

trap lines has been generated recently using transposable

elements (Sundaresan et al., 1995). Among these

lines are some that exhibit reporter gene expression in specific

female gametophyte cells, including the synergid cells,

the antipodal cells, and the central cell (Grossniklaus et al.,

1995). These lines will be extremely useful as markers for the

different female gametophyte cell types. For example, each

of these lines could be crossed with the existing group of

megagametogenesis mutants to determine whether the mutant

phenotypes are associated with inappropriate cell

specification. Furthermore, the trapped genes could serve

as entry points into the female gametophyte gene regulatory

circuitry.

Identify a gene that is specifically expressed in the female gametophyte, then isolate the promoter, fuse it to a GFP, then get transgenic lines. Find the best GFP expression line (specific transcriptional zone profile) then perform Fluorescence-Activated Cell Sorting. Get GFP+cells (gametophyte cells) then, extract DNA.