Analysis of the female gametophyte transcriptome of Arabidopsis by comparative expression profiling

Abstract

The extensive data on the transcription of the plant genome are derived primarily from the sporophytic generation. There currently is little information on genes that are expressed during female gametophyte development in angiosperms, and it is not known whether the female gametophyte transcriptome contains a major set of genes that are not expressed in the sporophyte or whether it is primarily a subset of the sporophytic transcriptome. Because the embryo sac is embedded within the maternal ovule tissue, we have utilized the Arabidopsis (Arabidopsis thaliana) mutant sporocyteless that produces ovules without embryo sacs, together with the ATH1 Arabidopsis whole-genome oligonucleotide array, to identify genes that are preferentially or specifically expressed in female gametophyte development. From analysis of the datasets, 225 genes are identified as female gametophyte genes, likely a lower limit as stringent criteria were used for the analysis, eliminating many low expressed genes. Nearly 45% of the identified genes were not previously detected by sporophytic expression profiling, suggesting that the embryo sac transcriptome may contain a significant fraction of transcripts restricted to the gametophyte. Validation of six candidate genes was performed using promoterbeta-glucuronidase fusions, and all of these showed embryo sac-specific expression in the ovule. The unfiltered expression data from this study can be used to evaluate the possibility of female gametophytic expression for any gene in the ATH1 array, and contribute to identification of the functions of the component of the Arabidopsis genome not represented in studies of sporophytic expression and function.

Figure 1.

Comparison of a homozygous spl mutant ovule with sibling heterozygous ovule, both at floral stage 13. A, spl mutant ovule showing the complete absence of the embryo sac but presence of all maternal cell types. B, Heterozygous spl plant ovule showing mature embryo sac. Abbreviations: cn, central cell nuclei; en, egg cell nucleus; et, endothelium; ii, inner integument; n, nucellus; oi, outer integument; sn, synergid nucleus. Scale bars represent 50 μm.

Figure 2.

Identified genes expressed during female gametophyte development. The gene sets included in the Venn diagrams were ≥2-fold higher in wild type than in spl with P values of <0.005 and signal values of >60.

Figure 3.

Distribution and classification of the female gametophyte genes.

Figure 4.

GUS expression patterns of the promoter∷GUS fusion with female gametophyte genes in early staged ovule. A to D, Ovule of wild-type plant at FG1 (A), FG2 (B), FG3 (C), and FG4 (D). A, The arrow points the nucleus of functional megaspore. B, Each arrow points a micropylar nucleus or chalazal nucleus within embryo sac. C, The two red arrows point the micropylar nuclei or chalazal nucleus, and a black arrow points a large central vacuole (cv). D, Embryo sac has two micropylar nuclei (red arrows), two chalazal nuclei (black arrows), and a large central vacuole (cv). E, The GUS activity of the promoter∷GUS fusion with At1g26795 was detected in embryo sac at FG4. F, GUS expression of the promoter∷GUS fusion with At1g36340 was detected in the closest nucleus to the chalazal end of the embryo sac at FG4. G, GUS activity of the promoter∷GUS fusion with At2g20070 was detected in dividing nuclei of the FG1 and FG2 embryo sac stages under the dark-field microscope. H, GUS expression of the promoter∷GUS fusion with At4g22050 was observed in chalazal end of embryo sac at FG4. I, GUS activity of the promoter∷GUS fusion with At5g40260 was detected in embryo sac at FG2. Scale bars represent 50 μm.

Figure 5.

GUS expression patterns of the promoter∷GUS fusion with female gametophyte genes in late staged ovule. A to C, Ovule of wild-type plant at FG5 (A), FG6 (B), and FG7 (C). D, GUS expression of the promoter∷GUS fusion with At1g26795 was detected in the embryo sac at FG5. E, GUS activity of the promoter∷GUS fusion with At1g36340 was detected in the antipodal cells at FG7. F and G, GUS expression of the promoter∷GUS fusion with At2g20070 (F) or At4g22050 (G) was observed in the chalazal end of the embryo sac at FG6. H, GUS activity of the promoter∷GUS fusion with At5g40260 was detected in the embryo sac at FG6. Abbreviations: an, antipodal cells; cn, central cell nucleus; en, egg cell nucleus; pn, polar nuclei; sn, synergid nuclei. Scale bars represent 50 μm.