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 promoter∷β-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.

The plant life cycle alternates between a haploid gametophytic phase and a diploid sporophytic phase. In angiosperms, gametophyte development takes place within the sporophyte. While the mature male gametophyte, pollen grains, are released from the anthers, the mature female gametophyte, also called embryo sac or megagametophyte, remains embedded within the maternal ovule tissues, making it difficult to access the female gametophyte for study. In Arabidopsis (Arabidopsis thaliana), the female gametophyte is a seven-cell structure consisting of four cell types: three antipodal cells, two synergid cells, one egg cell, and one central cell. Relatively little information is available on the genes expressed in the female gametophyte, since embryo sacs are small and it is difficult to isolate them free of maternal tissue contamination.

Recently, we identified 130 female gametophytic mutants from a population of insertional mutants that were analyzed in their genetic and molecular characteristics (Pagnussat et al., 2005). However, it is estimated that several thousand genes could be required for female gametophyte development (for review, see Drews and Yadegari, 2002), and alternate strategies are needed to identify additional genes involved in this developmental process. In animals, the oocyte transcriptome has been studied in humans, Caenorhabditis elegans, mice, bovine, and rainbow trout using cDNA or oligonucleotide arrays (Miller et al., 2003; Dobson et al., 2004; Hamatani et al., 2004; Yao et al., 2004; von Schalburg et al., 2005). In Arabidopsis, the Affymetrix ATH1 oligonucleotide chip, which contains probe sets for 22,591 annotated genes, has been used to study expression profiles at the whole-genome level. While genomic expression profiles have been used to identify genes related to reproduction, including seed development, floral organs (petal, sepal, stamen, and carpel), and male gametophytes by cDNA, oligonucleotide arrays (Honys and Twell, 2003, 2004; Köhler et al., 2003; Hennig et al., 2004; Wellmer et al., 2004), or peptide sequencing (Mayfield et al., 2001), a study of the female gametophyte transcriptome has not been performed at the whole-genome level.

We have previously described a gene called SPOROCYTELESS (SPL), which we showed to be required for initiation of both microsporogenesis and megasporogenesis in Arabidopsis (Yang et al., 1999). The SPL gene, which is identical to the NOZZLE gene (Schiefthaler et al., 1999), encodes a putative transcription factor expressed in the sporogenous cells of the anther and the ovule. Overexpression of SPL in wild-type flowers has no phenotype (Yang et al., 1999; Ito et al., 2004), but in agamous mutant flowers ectopic SPL can induce sporogenesis in petals (Ito et al., 2004). In this study, we identify genes involved in female gametophyte development through gene expression by comparison of wild-type ovules (embryo sac⁺) versus spl mutant ovules (embryo sac⁻) at the whole-genome level using the Affymetrix ATH1 oligonucleotide array. In addition, we determined the expression patterns of selected genes using promoter∷β-glucuronidase (GUS) fusions and found that the predictions based on the microarray analysis are in good agreement with the actual expression patterns. Our study provides a dataset of genes that are likely to be specific or preferentially expressed components of the female gametophyte transcriptome.

RESULTS

Sample Preparation to Analyze the Transcripts Related to Female Gametophyte Development

To characterize genes involved in embryo sac development, we set out to identify the genes having specific or increased expression in the ovules of heterozygous siblings (spl/SPL) compared to those of homozygous spl mutants (spl/spl) using the Affymetrix ATH1 oligonucleotide array. The spl mutation was identified by its complete male and female sterility as a single recessive mutation (Yang et al., 1999). The megasporocyte development in ovules of homozygous spl mutants is arrested at the archesporial cell and fails to undergo meiosis. Although megasporocytes are not formed and nucellus are arrested until the completion of integuments development, both inner and outer integuments and endothelium differentiated normally as in wild-type ovules (Fig. 1). Two ovule samples were collected according to different floral stages (Table I). The “early staged ovule” sample was collected from flowers of late 11 stage that have green anthers, and petals and long stamens of the same length, and mid-12 stage that have yellow anthers and in which the length of petal is longer than that of long stamen. The “late staged ovule” sample was obtained from flowers of late 12 stage that show protruded stigma from green bud, and 13 stage that have white bending petals and in which the length of stigma and long stamen is the same (Bowman, 1994). Additional precautions were taken when late staged ovules of heterozygous plants were collected to avoid pistil pollination. We detached anthers from flowers of early 12 stage and collected ovules after the flowers reached the appropriate floral stages (late 12 to 13). The collected ovules of each sample were randomly cleared, and the embryo sac stages were confirmed under the differential interference contrast microscope. The early staged ovules from heterozygotes contained functional megaspores or two- or four-nucleated embryo sacs (FG1–FG4), and the late staged ovules contained eight-nucleated embryo sacs or mature embryo sacs (FG5–FG7). The experiments were performed in triplicate. The three samples of each stage consisting of about 2,500 to approximately 3,000 ovules were independently collected from at least six different plants to minimize any effects of plant-to-plant transcriptional variations, and labeled RNAs made from each sample were hybridized separately with an Affymetrix ATH1 array. These ovule populations were enough to make the RNA probe for direct microarray hybridization without any amplification step. In the case of late staged ovules, we prepared two additional spl ovule samples from spl emasculated flowers to verify that detaching the anthers would not significantly affect the expression profile of the ovules.

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.

Table I.

The ovule sample collected on the basis of stages of flowers and embryo sacs

Sample Division	Floral Stagea	Embryo Sac Stageb
Early staged ovule	Late 11	FG1 (one nucleate)
	Early 12	FG2 (early two nucleate)
		FG3 (late two nucleate)
	Mid 12	FG4 (four nucleate)
Late staged ovule	Late 12	FG5 (eight nucleate)
	13	FG6 (seven celled)
		FG7 (four celled)

^aFloral stages are described by Bowman (1994).

^bEmbryo sac stages are summarized from Christensen et al. (1997).

Statistical Data Analysis of the Genes Involved in Female Gametophyte Development

Arabidopsis ATH1 whole-genome arrays, which contain oligos for 24,000 Arabidopsis gene sequences, were used to study comparative gene expressions during embryo sac development of heterozygous siblings (spl/SPL) and spl mutants (spl/spl). Signal intensities were calculated by a perfect match (PM)-only model (Li and Wong, 2001a) that uses only PM probes to calculate all-positive expression values as a statistical algorithm using the dChip program (version 1.3) freely available at http://www.dchip.org/ (Li and Wong, 2001b). The median intensities and Present call% obtained by the dChip program were used to assess the overall quality of the arrays or differences between arrays, and hybridization controls such as bioB, bioC, bioD, and cre and internal control genes such as actin and GAPDH were used to evaluate sample hybridization efficiency to gene expression arrays and RNA sample quality. As a result, all analyzed arrays were judged to have high quality (data not shown).

We first calculated the correlation coefficient to ensure the reliability and determine the reproducibility of the microarray analysis using average difference based on the signal intensities between arrays using statistic package R program (version 1.6.1; http://www.r-project.org/; Ihaka and Gentleman, 1996). All 14 arrays had high correlation coefficients of >0.94, which suggested an excellent reproducibility among individual samples. Especially strong positive correlation between samples of the same kind was demonstrated with the correlation coefficients of ≥0.98 (Supplemental Table I).

The signal values of data were not normalized, and these raw signal data were used for data analysis without any filtering methods, such as corrections for between-chip heterogeneity and eliminations of backgrounds, because we did not want to remove potential genes of interest (Thomas et al., 2001). The following three screening criteria were applied for data analysis.

First, we applied the conventional t test to the raw signal data. This t test provides the probability (P) that a difference in gene expression occurred by chance. The P values were calculated by Student's t test for two-sample equal variance (homoscedastic; Devore and Peck, 1997; Pan, 2002). The genes were judged significantly changed when they were assigned a P value of <0.005. The signal values from ovules of spl mutants were used as a baseline. In late staged ovule data, signal values of emasculated spl and that of nonemasculated spl were combined and used as signal values of spl mutants at late stage. A total of 508 genes at early staged ovule and 1,015 genes at late staged ovule satisfied this criterion.

Second, we applied a 2-fold cutoff for the genes with <0.005 P value and retained only the genes that showed 2-fold or greater expression in heterozygous ovules (embryo sac⁺) as compared to spl mutant ovules (embryo sac⁻). According to these criteria, 107 genes at early staged ovule and 248 genes at late staged ovule were identified.

Third, genes with hybridization signals of <60 in heterozygous ovules were removed on the basis of signal intensities of the poly-A controls (dap, lys, phe, thr, trp) of Affymetrix ATH1.

When we applied all three criteria, 23 genes were identified in early staged ovules, 128 genes in late staged ovules, and 74 genes in both early and late staged ovules (Fig. 2). We also compared the signal intensities of late staged nonemasculated (Supplemental Fig. 1) spl ovules with late staged wild-type ovules. We found that the results from this comparison were similar to the comparison of the combined late staged emasculated and nonemasculated spl ovules with the late staged wild-type ovules, indicating that emasculation did not significantly affect the outcome (Fig. 2). The whole data set for 14 arrays is available at the laboratory Web site (http://sundarlab.ucdavis.edu/) and in the supplemental data.

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.

Functional Classification and Identification of the Embryo Sac Genes

We classified functionally the 225 genes that are predicted to be expressed during megagametogenesis on the basis of the biological or biochemical function of the gene ontology annotation for the Arabidopsis genome provided by The Arabidopsis Information Resource at www.arabidopsis.org. Using the current annotation, 22 (9.8%) of the 225 genes encode predicted hypothetical proteins, and 53 genes (23.6%) encode proteins with an expressed sequence tag match but without any protein match (unknown proteins) or unclassified proteins as shown in Figure 3. These numbers indicate an overrepresentation of the classes of unknown and hypothetical proteins, representing approximately 33% of the female gametophyte genes in this study, as compared to approximately 21% for the whole Arabidopsis genome. The remaining genes were distributed across all the major classification groups from central metabolism, detoxification/stress response, cell structure organization, and transport, to protein degradation, signal transduction, and transcriptional regulation. The detailed gene information for genes within each classification group is provided in Table II.

Figure 3.

Distribution and classification of the female gametophyte genes.

Table II.

Identification of embryo sac genes

Genes were selected on the basis of the P value of <0.0050, fold change of >2.0, and signal intensity of >60.0 at wild-type siblings. WT^1–3, Mean of signal intensities for arrays 1 to 3 of wild-type siblings. spl^1–3, Mean of signal intensities for arrays 1 to 3 of spl mutants. WT^d1–d3, Mean of signal intensities for arrays 1 to 3 of emasculated wild-type siblings. spl^{1–3, d1–d2}, Mean of signal intensities for arrays 1 to 3 of nonemasculated spl, and arrays 1 and 2 of emasculated spl.

Affymetrix Codea	Gene IDb	Description	Early Staged Ovule				Late Staged Ovule				Divisione
			WT1–3	spl1–3	FCc	P Valued	WTd1–d3	spl1–3, d1–d2	FCc	P Valued
Unknown protein
261271_at	At1g26795	Self-incompatibility protein related	769	30	25.3	0.000083	761	27	28.2	0.000000	Both
265133_s_at	At1g51250	Expressed protein	151	25	6.1	0.001456	539	19	28.2	0.000000	Both
259726_at	At1g60985	Expressed protein	98	16	5.9	0.002140	349	16	21.4	0.000003	Both
265762_at	At2g01240	Reticulon family protein (RTNLB15)	227	15	15.1	0.000418	148	14	10.3	0.000020	Both
263713_at	At2g20595	Expressed protein	278	15	18.9	0.003556	1,047	25	41.4	0.000000	Both
267193_at	At2g30900	Expressed protein	97	23	4.3	0.000554	108	20	5.4	0.000016	Both
256719_at	At2g34130	CACTA-like transposase family (Ptta/En/Spm)	117	11	10.3	0.000181	114	10	11.8	0.000000	Both
256600_at	At3g14850	Expressed protein	261	34	7.6	0.000088	294	35	8.5	0.000000	Both
257889_at	At3g17080	Self-incompatibility protein related	187	30	6.2	0.002931	342	19	17.8	0.000000	Both
251698_at	At3g56610	Expressed protein	518	16	32.0	0.004682	1,444	31	46.8	0.000000	Both
254257_s_at	At4g23350	Expressed protein	104	30	3.5	0.003879	335	22	15.3	0.000000	Both
253164_at	At4g35725	Expressed protein	158	15	10.4	0.003044	298	14	21.2	0.000024	Both
250871_at	At5g03930	Expressed protein	171	24	7.0	0.001971	106	14	7.5	0.000002	Both
250325_s_at	At5g12060	Self-incompatibility protein related	138	41	3.4	0.001255	206	30	6.8	0.000001	Both
249855_at	At5g22970	Expressed protein	261	25	10.6	0.003517	1,028	32	31.7	0.000001	Both
249757_at	At5g24316	Pro-rich family protein	471	17	27.7	0.001766	1,328	29	45.6	0.000000	Both
249401_at	At5g40260	Nodulin MtN3 family protein	566	37	15.2	0.000603	427	30	14.3	0.000004	Both
248284_at	At5g52975	Expressed protein	755	81	9.3	0.000353	403	49	8.2	0.000001	Both
262503_at	At1g21670	Expressed protein	161	41	4.0	0.000009	64	45	1.4	0.031616	Early
261731_s_at	At1g47780	Acyl-protein thioesterase related	84	15	5.6	0.004140	60	13	4.7	0.000003	Early
249375_at	At5g40730	Arabinogalactan protein (AGP24)	806	129	6.2	0.000342	661	575	1.1	0.245297	Early
264610_at	At1g04645	Self-incompatibility protein related	106	18	5.7	0.157950	793	21	38.1	0.000557	Late
261846_at	At1g11540	Expressed protein	145	107	1.4	0.117890	170	85	2.0	0.000012	Late
255908_s_at	At1g18010	Expressed protein	128	64	2.0	0.018058	93	45	2.1	0.000005	Late
256079_at	At1g20680	Expressed protein	56	14	3.9	0.011798	67	12	5.7	0.000019	Late
263027_at	At1g24010	Expressed protein	23	12	1.9	0.256580	96	22	4.3	0.000001	Late
260942_s_at	At1g45190	Expressed protein	318	6	49.3	0.007497	1,404	30	47.1	0.000000	Late
265138_at	At1g51300	Acyl-protein thioesterase related	192	43	4.4	0.015275	450	49	9.1	0.000000	Late
262314_at	At1g70810	C2 domain-containing protein	23	17	1.4	0.182775	65	17	3.8	0.000004	Late
262972_at	At1g75620	Glyoxal oxidase related	67	49	1.4	0.205253	133	38	3.5	0.000079	Late
264297_at	At1g78710	Expressed protein	120	62	1.9	0.005001	144	57	2.5	0.000001	Late
267241_at	At2g02490	Hydroxyproline-rich glycoprotein family protein	45	4	10.6	0.052466	395	11	37.0	0.000186	Late
267218_at	At2g02515	Expressed protein	163	39	4.2	0.007206	838	34	24.8	0.000001	Late
265517_at	At2g06090	Self-incompatibility protein related	254	14	17.8	0.013973	1,109	23	47.4	0.000000	Late
264590_at	At2g17710	Expressed protein	144	194	0.7	0.361347	841	394	2.1	0.000010	Late
263518_at	At2g21655	Expressed protein	271	17	15.8	0.015412	946	25	37.6	0.000001	Late
265674_at	At2g32190	Expressed protein	100	61	1.6	0.072973	198	70	2.8	0.000267	Late
265670_s_at	At2g32210	Expressed protein	155	158	1.0	0.888298	213	76	2.8	0.000186	Late
265245_at	At2g43060	Expressed protein	39	45	0.9	0.564913	131	62	2.1	0.000340	Late
259107_at	At3g05460	Sporozoite surface protein related	403	43	9.4	0.008348	1,415	46	31.1	0.000069	Late
258130_at	At3g24510	Expressed protein	486	10	48.6	0.012987	2,033	38	53.8	0.000000	Late
252253_at	At3g49300	Pro-rich family protein	251	42	6.0	0.007754	626	32	19.3	0.000044	Late
251606_at	At3g57840	Self-incompatibility protein related	109	39	2.8	0.058391	468	38	12.4	0.000005	Late
255207_at	At4g07515	Expressed protein	453	23	19.8	0.008275	859	32	27.0	0.000000	Late
245424_at	At4g17505	Expressed protein	157	55	2.8	0.005099	312	33	9.4	0.000003	Late
254494_at	At4g20050	Expressed protein	125	85	1.5	0.069310	230	59	3.9	0.000001	Late
254001_at	At4g26260	Expressed protein	174	62	2.8	0.067591	149	25	5.9	0.000073	Late
253724_at	At4g29285	Expressed protein	91	12	7.3	0.029036	647	16	39.5	0.000000	Late
253656_at	At4g30090	Expressed protein	101	85	1.2	0.276358	76	37	2.1	0.000137	Late
253401_at	At4g32870	Expressed protein	88	41	2.1	0.040870	165	39	4.3	0.000012	Late
246641_s_at	At5g34885	Expressed protein	216	21	10.3	0.025229	631	27	23.5	0.000000	Late
249179_at	At5g42955	Expressed protein	266	14	19.3	0.014861	882	23	38.0	0.000000	Late
248892_at	At5g46300	Expressed protein	62	5	11.8	0.014887	124	5	23.1	0.000001	Late
Central intermediary metabolism
259786_at	At1g29660	GDSL-motif lipase/hydrolase family protein	289	84	3.4	0.003166	879	425	2.1	0.000131	Both
260124_at	At1g36340	Ubiquitin-conjugating enzyme, E2	584	38	15.3	0.001661	467	34	13.8	0.000002	Both
245672_at	At1g56710	Glycoside hydrolase family 28 protein	218	85	2.6	0.004220	235	94	2.5	0.000298	Both
257442_at	At2g28680	Cupin family protein	316	47	6.8	0.001424	303	33	9.2	0.000008	Both
267408_at	At2g34890	CTP synthase, putative	152	25	6.1	0.000651	67	16	4.2	0.000011	Both
257243_at	At3g24230	Pectate lyase family protein	332	59	5.7	0.003792	198	63	3.2	0.000001	Both
258763_s_at	At3g30540	(1-4)-β-Mannan endohydrolase family	125	8	15.9	0.002254	148	9	17.4	0.000000	Both
252342_at	At3g48950	Glycoside hydrolase family 28 protein	348	30	11.6	0.001576	603	26	23.3	0.000000	Both
248925_at	At5g45910	GDSL-motif lipase/hydrolase-like protein	279	14	20.0	0.001613	1,367	26	53.5	0.000000	Both
247228_at	At5g65140	Trehalose-6-phosphate phosphatase	305	38	8.0	0.000647	246	92	2.7	0.000044	Both
264147_at	At1g02200	CER1 protein	206	85	2.4	0.000205	40	39	1.0	0.799875	Early
264146_at	At1g02205	CER1 protein, At1g02200	650	279	2.3	0.000021	219	225	1.0	0.820363	Early
259703_at	At1g77790	Glycosyl hydrolase family 17 protein	113	16	7.0	0.000428	54	11	4.8	0.000000	Early
267202_s_at	At2g31030	Oxysterol-binding family protein	74	10	7.3	0.000864	27	7	3.8	0.000005	Early
260611_at	At2g43670	Glycosyl hydrolase family 17 protein	132	53	2.5	0.002326	129	75	1.7	0.000212	Early
252320_at	At3g48580	Xyloglucan:xyloglucosyl transferase, putative	743	315	2.4	0.003795	1,176	766	1.5	0.004917	Early
250082_at	At5g17200	Glycoside hydrolase family 28 protein	71	17	4.2	0.002910	20	11	1.8	0.000578	Early
260947_at	At1g06020	PfkB-type carbohydrate kinase family protein	58	40	1.5	0.010463	75	29	2.6	0.000134	Late
259391_s_at	At1g06350	Fatty acid desaturase family protein	163	98	1.7	0.342723	196	88	2.2	0.000376	Late
255956_at	At1g22015	Galactosyltransferase family protein	158	43	3.7	0.007113	274	34	8.0	0.000000	Late
245792_at	At1g32100	Pinoresinol-lariciresinol reductase, putative	66	66	1.0	0.993453	184	68	2.7	0.000127	Late
245794_at	At1g32170	Xyloglucan:xyloglucosyl transferase, putative	59	49	1.2	0.363717	101	47	2.1	0.000150	Late
256211_at	At1g50960	Gibberellin 20-oxidase related	42	19	2.2	0.015340	75	14	5.4	0.000000	Late
260333_at	At1g70500	Polygalacturonase, putative/pectinase, putative	581	27	21.7	0.007959	674	42	16.0	0.000001	Late
260066_at	At1g73610	GDSL-motif lipase/hydrolase family protein	69	24	2.9	0.035163	163	20	8.0	0.000056	Late
260259_at	At1g74300	Esterase/lipase/thioesterase family protein	60	57	1.1	0.690243	105	47	2.2	0.000095	Late
265331_at	At2g18420	Gibberellin-responsive protein, putative	27	25	1.1	0.777953	73	18	4.1	0.000139	Late
267607_s_at	At2g26740	Epoxide hydrolase, soluble (sEH)	138	137	1.0	0.977676	205	87	2.4	0.000242	Late
267337_at	At2g39980	Transferase family protein	93	46	2.0	0.015251	69	30	2.3	0.000006	Late
260559_at	At2g43860	Polygalacturonase, putative/pectinase, putative	113	38	3.0	0.005920	69	30	2.3	0.000032	Late
258767_at	At3g10890	(1-4)-β-Mannan endohydrolase, putative	100	16	6.3	0.013753	698	20	35.1	0.000002	Late
258151_at	At3g18080	Glycosyl hydrolase family 1 protein	199	194	1.0	0.899935	384	124	3.1	0.000003	Late
257065_at	At3g18220	Phosphatidic acid phosphatase family protein	45	29	1.5	0.034020	63	22	2.8	0.000003	Late
251491_at	At3g59480	PfkB-type carbohydrate kinase family protein	549	56	9.9	0.032782	598	210	2.8	0.001882	Late
255550_at	At4g01970	Galactinol-raffinose galactosyltransferase, putative	127	41	3.1	0.006794	226	83	2.7	0.000170	Late
245349_at	At4g16690	Esterase/lipase/thioesterase family protein	75	40	1.9	0.193063	112	53	2.1	0.000368	Late
254609_at	At4g18970	GDSL-motif lipase/hydrolase family protein	461	508	0.9	0.189942	703	309	2.3	0.000085	Late
246498_at	At5g16230	Acyl-[acyl-carrier-protein] desaturase, putative	34	26	1.3	0.219929	84	23	3.6	0.001229	Late
249983_at	At5g18470	Curculin-like (mannose-binding) lectin family protein	44	35	1.2	0.486888	106	48	2.2	0.001507	Late
246774_at	At5g27530	Glycoside hydrolase family 28 protein	108	46	2.4	0.012277	146	31	4.8	0.000001	Late
249474_s_at	At5g39190	Germin-like protein (GER2)	14	12	1.2	0.500732	99	13	7.6	0.000004	Late
248812_at	At5g47330	Palmitoyl protein thioesterase family protein	63	78	0.8	0.143704	203	43	4.7	0.000004	Late
248791_at	At5g47350	Palmitoyl protein thioesterase family protein	212	65	3.3	0.040644	1,167	291	4.0	0.000003	Late
Hypothetical protein
260318_at	At1g63960	Hypothetical protein	62	20	3.2	0.002924	72	20	3.5	0.000014	Both
265579_at	At2g20070	Hypothetical protein	661	48	13.8	0.000041	487	36	13.4	0.000000	Both
257434_at	At2g21740	Hypothetical protein	128	25	5.0	0.001238	301	20	15.3	0.000000	Both
252753_at	At3g43500	Hypothetical protein	80	20	4.0	0.002002	131	20	6.6	0.000002	Both
246859_at	At5g25950	Hypothetical protein	70	13	5.4	0.000045	131	12	10.9	0.000000	Both
248225_at	At5g53740	Hypothetical protein	168	72	2.3	0.000851	111	52	2.1	0.000065	Both
266706_at	At2g03320	Hypothetical protein	97	28	3.5	0.001116	47	17	2.8	0.000175	Early
246866_at	At5g25960	Hypothetical protein	131	11	11.7	0.000000	41	9	4.8	0.000020	Early
248396_at	At5g52130	Hypothetical protein	111	21	5.4	0.000609	47	15	3.2	0.000216	Early
257468_at	At1g47470	Hypothetical protein	147	13	11.2	0.029273	1,010	25	40.1	0.000000	Late
261313_at	At1g52970	Hypothetical protein	484	49	9.9	0.007905	1,881	59	32.1	0.000000	Late
259944_at	At1g71470	Hypothetical protein	72	60	1.2	0.213121	71	33	2.1	0.000104	Late
263895_at	At2g21920	Hypothetical protein	107	26	4.2	0.007542	73	17	4.2	0.000906	Late
258798_at	At3g04540	Hypothetical protein	202	55	3.7	0.087047	1,542	50	30.9	0.000000	Late
256773_at	At3g13630	Hypothetical protein	31	28	1.1	0.715677	60	27	2.2	0.000839	Late
251607_at	At3g57850	Hypothetical protein	73	11	6.6	0.036056	370	13	29.4	0.000050	Late
255029_x_at	At4g09470	Hypothetical protein	139	8	17.2	0.012714	687	16	43.5	0.000003	Late
255804_at	At4g10220	Hypothetical protein	120	31	3.9	0.009652	410	34	12.0	0.000003	Late
254692_at	At4g17860	Hypothetical protein	60	35	1.7	0.028983	107	28	3.9	0.000008	Late
246472_at	At5g17130	Hypothetical protein	80	83	1.0	0.853233	75	34	2.2	0.000487	Late
249157_at	At5g43510	Hypothetical protein	356	13	27.3	0.012178	588	29	20.1	0.000001	Late
247245_at	At5g64720	Hypothetical protein	105	15	6.9	0.010392	322	13	24.5	0.000000	Late
Detoxification/stress response
264001_at	At2g22420	Peroxidase 17 (PER17)	791	121	6.5	0.003718	725	120	6.1	0.000007	Both
265264_at	At2g42930	Glycosyl hydrolase family protein 17	346	55	6.3	0.003492	285	34	8.5	0.000007	Both
254000_at	At4g26250	Galactinol synthase induced by water stress	89	18	4.8	0.003892	65	15	4.2	0.000004	Both
252951_at	At4g38700	Disease resistance-responsive family protein	248	109	2.3	0.004228	706	115	6.2	0.000000	Both
247857_at	At5g58400	Peroxidase, putative	1,053	240	4.4	0.000004	473	161	2.9	0.000023	Both
261410_at	At1g07610	Metallothionein-like protein 1C (MT-1C)	333	159	2.1	0.000832	238	170	1.4	0.168590	Early
266743_at	At2g02990	Ribonuclease 1 (RNS1)	67	23	2.9	0.001354	140	100	1.4	0.201557	Early
263026_at	At1g24000	Bet v I allergen family protein	43	15	2.9	0.266278	250	19	12.9	0.000001	Late
265920_s_at	At2g15120	Pseudogene, disease-resistance family protein	38	33	1.1	0.299880	103	23	4.6	0.000933	Late
266562_at	At2g23970	Defense-related protein, putative	68	60	1.1	0.479766	194	38	5.1	0.000000	Late
267138_s_at	At2g38210	Ethylene-responsive protein, putative	181	86	2.1	0.091609	246	76	3.2	0.001233	Late
266169_at	At2g38900	Ser protease inhibitor	40	37	1.1	0.867313	343	105	3.3	0.000002	Late
260557_at	At2g43610	Glycoside hydrolase family 19 protein	94	49	1.9	0.003261	76	33	2.3	0.000002	Late
258791_at	At3g04720	Hevein-like protein (HEL)	35	35	1.0	0.937369	91	34	2.6	0.003414	Late
258172_at	At3g21620	Early responsive to dehydration protein related	60	24	2.5	0.000597	68	21	3.2	0.000002	Late
254098_at	At4g25100	Superoxide dismutase (Fe), chloroplast (SODB)	688	313	2.2	0.060518	76	14	5.6	0.000220	Late
253655_at	At4g30070	Plant defensin-fusion protein, putative	99	39	2.5	0.007716	167	23	7.4	0.000001	Late
250200_at	At5g14130	Peroxidase, putative	94	67	1.4	0.229669	106	41	2.6	0.000012	Late
250083_at	At5g17220	Glutathione S-transferase-like protein	99	93	1.1	0.870900	537	235	2.3	0.000020	Late
249560_at	At5g38330	Plant defensin-fusion protein, putative	280	26	11.0	0.008435	1,140	29	39.5	0.000000	Late
249527_at	At5g38710	Pro oxidase, putative	70	18	3.9	0.006176	74	15	4.8	0.000019	Late
Cell structure organization
260573_at	At2g47280	Pectinesterase family protein	227	36	6.4	0.000454	139	25	5.5	0.000000	Both
257878_at	At3g17150	Invertase/pectin methylesterase inhibitor family	399	26	15.3	0.004375	1,121	38	29.2	0.000000	Both
258438_at	At3g17230	Invertase/pectin methylesterase inhibitor family	434	30	14.4	0.000924	758	30	24.8	0.000000	Both
251748_at	At3g55680	Invertase/pectin methylesterase inhibitor family	162	51	3.2	0.000901	112	39	2.8	0.000002	Both
255699_at	At4g00190	Putative pectinesterase	86	31	2.8	0.001537	90	24	3.7	0.000001	Both
248823_s_at	At5g46960	Invertase/pectin methylesterase inhibitor family	270	8	35.1	0.002890	519	13	39.4	0.000003	Both
247246_at	At5g64620	Invertase/pectin methylesterase inhibitor family	747	338	2.2	0.000298	870	425	2.0	0.000000	Both
260802_at	At1g78400	Glycoside hydrolase family 28 protein	201	23	8.8	0.000001	30	18	1.7	0.001391	Early
258764_at	At3g10720	Pectinesterase, putative	415	205	2.0	0.001974	112	77	1.5	0.008263	Early
253725_at	At4g29340	Profilin 3 (PRO3) (PFN3)	63	27	2.3	0.000446	26	23	1.1	0.244366	Early
264500_at	At1g09370	Pectinesterase inhibitor domain-containing protein	61	10	6.0	0.123949	594	14	43.8	0.000012	Late
259613_at	At1g48010	Invertase/pectin methylesterase inhibitor family	38	36	1.1	0.597625	105	29	3.6	0.000000	Late
262083_at	At1g56100	Pectinesterase inhibitor domain-containing protein	128	53	2.4	0.444629	2,622	746	3.5	0.000012	Late
245656_at	At1g56620	Pectinesterase inhibitor domain-containing protein	50	26	1.9	0.044275	252	19	13.3	0.000000	Late
257679_at	At3g20470	Pseudogene, Gly-rich protein	117	99	1.2	0.700308	578	267	2.2	0.001050	Late
245371_at	At4g15750	Invertase/pectin methylesterase inhibitor family	200	63	3.2	0.067454	1,378	440	3.1	0.000004	Late
249962_at	At5g18990	Pectinesterase family protein	39	19	2.0	0.028245	82	15	5.5	0.000041	Late
247377_at	At5g63180	Pectate lyase family protein	175	125	1.4	0.147262	410	192	2.1	0.000518	Late
Transport
260319_at	At1g63950	Heavy-metal-associated domain-containing protein	310	28	10.9	0.002905	148	19	7.9	0.000001	Both
259757_at	At1g77510	Protein disulfide isomerase, putative	1,163	447	2.6	0.000344	943	367	2.6	0.000170	Both
258760_at	At3g10780	Emp24/gp25L/p24 family protein	435	94	4.6	0.001770	400	75	5.4	0.000000	Both
245892_at	At5g09370	Protease inhibitor/seed storage/lipid transfer protein	384	57	6.8	0.004630	1,326	108	12.3	0.000002	Both
263765_at	At2g21540	SEC14 cytosolic factor, putative	135	64	2.1	0.000985	64	42	1.5	0.002042	Early
249346_at	At5g40780	Lys- and His-specific transporter, putative	783	170	4.6	0.002599	877	624	1.4	0.054790	Early
264520_at	At1g10010	Amino acid permease, putative	91	35	2.6	0.005939	167	34	4.8	0.000000	Late
265002_at	At1g24400	Lys- and His-specific transporter	156	41	3.8	0.024933	311	142	2.2	0.000005	Late
259580_at	At1g28030	Oxidoreductase, 2OG-Fe(II) oxygenase family protein	52	44	1.2	0.276557	95	32	3.0	0.000024	Late
265064_at	At1g61630	Equilibrative nucleoside transporter, putative (ENT7)	70	40	1.7	0.013220	67	29	2.3	0.000179	Late
259844_at	At1g73560	Protease inhibitor/seed storage/lipid transfer protein	49	22	2.2	0.005234	91	19	4.9	0.000122	Late
264482_at	At1g77210	Sugar transporter, putative	73	65	1.1	0.601658	118	55	2.1	0.000012	Late
257366_s_at	At2g03040	Transmembrane protein related	159	39	4.1	0.005632	184	22	8.3	0.000008	Late
266276_at	At2g29330	Tropinone reductase, putative	68	30	2.3	0.007410	68	28	2.4	0.000243	Late
254453_at	At4g21120	Amino acid permease family protein	211	64	3.3	0.007991	259	88	2.9	0.000000	Late
246887_at	At5g26250	Sugar transporter, putative	159	61	2.6	0.015475	146	37	4.0	0.000013	Late
248275_at	At5g53520	Oligopeptide transporter OPT family protein	43	32	1.3	0.204722	80	20	3.9	0.000002	Late
248019_at	At5g56480	Protease inhibitor/seed storage/lipid transfer protein	61	50	1.2	0.206709	111	38	2.9	0.000015	Late
Protein degradation
256486_at	At1g31450	Aspartyl protease family protein	263	18	14.8	0.001311	607	17	35.1	0.000000	Both
245738_at	At1g44130	Nucellin protein, putative	117	10	12.3	0.002988	94	8	11.2	0.000067	Both
259368_at	At1g69100	Aspartyl protease family protein	546	13	42.6	0.003756	952	33	29.1	0.000000	Both
252499_s_at	At3g46840	Subtilase family protein	389	34	11.6	0.000398	443	26	17.2	0.000001	Both
245589_at	At4g15040	Subtilase family protein	171	23	7.3	0.000154	66	17	4.0	0.000055	Both
254336_at	At4g22050	Aspartyl protease family protein	681	37	18.3	0.000096	714	67	10.6	0.000000	Both
246684_at	At5g33340	Aspartyl protease family protein	98	25	4.0	0.000228	189	21	9.1	0.000001	Both
247798_at	At5g58830	Subtilase family protein	73	15	5.0	0.002673	235	13	17.7	0.000000	Both
254237_at	At4g23520	Cys proteinase, putative	67	28	2.4	0.000243	41	22	1.9	0.000254	Early
247697_at	At5g59810	Subtilase family protein	92	43	2.1	0.002288	56	44	1.3	0.003872	Early
264067_x_at	At2g28010	Aspartyl protease family protein	33	19	1.7	0.039844	78	13	5.9	0.000004	Late
250345_at	At5g11940	Subtilase family protein	58	15	3.9	0.005591	154	14	11.4	0.000000	Late
Signal transduction
263740_at	At2g20660	Rapid alkalinization factor (RALF) family protein	82	33	2.4	0.000758	90	24	3.8	0.000000	Both
245158_at	At2g33130	RALF family protein	435	34	12.8	0.000989	437	38	11.4	0.000000	Both
266418_at	At2g38750	Annexin 4 (ANN4)	274	62	4.4	0.003825	220	67	3.3	0.000001	Both
251514_at	At3g59260	Pirin, putative	202	33	6.1	0.001924	171	30	5.7	0.000002	Both
255489_at	At4g02650	Epsin N-terminal homology domain-containing protein	448	22	20.7	0.004357	1,286	32	39.8	0.000000	Both
245177_at	At5g12380	Annexin, putative	190	17	11.2	0.000393	215	17	12.8	0.000000	Both
249013_at	At5g44700	Leu-rich repeat transmembrane protein kinase	81	31	2.6	0.001303	75	30	2.5	0.000305	Both
261285_at	At1g35720	Annexin 1 (ANN1)	168	122	1.4	0.273697	393	108	3.6	0.000012	Late
257869_at	At3g25160	ER lumen protein retaining receptor family protein	86	51	1.7	0.030431	147	52	2.8	0.000020	Late
250090_at	At5g17330	Glutamate decarboxylase 1 (GAD 1)	195	21	9.2	0.005864	222	25	9.0	0.000000	Late
Energy metabolism
259268_at	At3g01070	Plastocyanin-like domain-containing protein	79	28	2.8	0.002166	126	26	4.8	0.000001	Both
253634_at	At4g30590	Plastocyanin-like domain-containing protein	705	56	12.6	0.000057	500	41	12.3	0.000005	Both
252897_at	At4g39490	Cytochrome P450 family protein, At4g38480	62	19	3.4	0.004614	134	18	7.5	0.000000	Both
248236_at	At5g53870	Plastocyanin-like domain-containing protein	1,236	60	20.7	0.000394	653	165	4.0	0.000009	Both
262528_at	At1g17260	ATPase 10, plasma membrane type, putative	45	26	1.7	0.385722	344	102	3.4	0.000002	Late
261396_at	At1g79800	Plastocyanin-like domain-containing protein	49	40	1.2	0.236124	69	31	2.2	0.000064	Late
266563_at	At2g23990	Plastocyanin-like domain-containing protein	195	47	4.2	0.011017	953	41	23.3	0.000015	Late
255690_at	At4g00360	Cytochrome P450, putative	49	23	2.1	0.018309	79	29	2.7	0.000026	Late
254489_at	At4g20800	FAD-binding domain-containing protein	47	11	4.4	0.027803	121	11	11.3	0.000000	Late
Transcriptional regulation
267528_at	At2g45650	MADS-box protein (AGL6)	245	59	4.2	0.002904	359	170	2.1	0.000048	Both
249338_at	At5g41090	No apical meristem (NAM) family protein	166	18	9.1	0.000764	77	13	5.8	0.000000	Both
260212_at	At1g74480	RWP-RK domain-containing protein	55	36	1.5	0.071151	65	29	2.2	0.001510	Late
266969_at	At2g39540	Gibberellin-regulated family protein	41	38	1.1	0.593532	179	32	5.6	0.000003	Late
254619_at	At4g18770	Myb family transcription factor (MYB98)	109	59	1.8	0.063530	110	41	2.7	0.000002	Late
251114_at	At5g01380	Expressed protein	25	26	1.0	0.855505	62	23	2.7	0.000211	Late
248240_at	At5g53950	No apical meristem (NAM) family protein	224	201	1.1	0.399544	269	110	2.4	0.000013	Late
Secondary metabolism
260386_at	At1g74010	Strictosidine synthase family protein	110	21	5.1	0.001313	368	22	16.4	0.000010	Both
264401_at	At1g61720	Dihydroflavonol 4-reductase family (BAN)	54	45	1.2	0.693582	535	176	3.0	0.000017	Late
260335_at	At1g74000	Strictosidine synthase family protein	144	72	2.0	0.005947	104	39	2.7	0.000038	Late
254283_s_at	At4g22870	Leucoanthocyanidin dioxygenase, putative	81	85	1.0	0.880918	416	183	2.3	0.000118	Late
249215_at	At5g42800	Dihydroflavonol 4-reductase	31	26	1.2	0.546725	301	84	3.6	0.000030	Late
Plant development/organogenesis
262113_at	At1g02820	Late embryogenesis abundant 3 family protein	82	33	2.5	0.000057	76	27	2.8	0.000001	Both
262659_at	At1g14240	Nucleoside phosphatase family protein	80	25	3.3	0.000911	92	16	5.8	0.000000	Both
262549_at	At1g31290	PAZ domain-containing protein	140	99	1.4	0.096043	169	63	2.7	0.000011	Late
252234_at	At3g49780	Phytosulfokines 3 (PSK3)	17	9	2.0	0.386279	163	43	3.8	0.000014	Late
251301_at	At3g61880	Cytochrome P450, putative	94	51	1.9	0.003809	91	41	2.2	0.000032	Late
Protein biosynthesis/DNA catabolism
245883_at	At5g09500	40S ribosomal protein S15 (RPS15C)	85	18	4.7	0.002080	54	9	5.9	0.000028	Early
260438_at	At1g68290	Bifunctional nuclease, putative	44	28	1.6	0.093917	149	25	5.9	0.000104	Late

^aAffymetrix probe set number.

^bArabidopsis Genome Initiative number.

^cFold change for signal intensity of wild type/signal intensity of spl.

^dP values calculated by Student's t test for two-sample equal variance.

^eExpressed ovule stage.

Promoter∷GUS Fusions Validate Embryo Sac-Specific Expression in Both Early and Late Staged Ovules

For validation of the microarray analysis results, we examined previously uncharacterized genes from 225 identified embryo sac genes. Six genes were selected to examine their expression pattern during female gametophyte development. To increase the probability of obtaining detectable levels of reporter gene expression in promoter fusion studies, two criteria were used. The first is that they rank in the top 20 when they are arranged by highest signal intensity of early staged wild-type ovule. The second is that they had a greater than 10-fold change for both early and late staged wild-type ovules compared to spl ovules (Table III). When we examined their RNA expression patterns using reverse transcription-PCR, all six genes were detected only in wild-type ovules of both of early and late stage and not in spl mutant ovules (data not shown). Next, we constructed promoter∷GUS cassettes to analyze their expression. All six promoter∷GUS fusions were examined during embryo sac development to determine whether they are expressed only in the embryo sac as predicted. We examined six transgenic lines for each promoter fusion, by analyzing five or more T2 generation plants for each transgenic line to ensure that the expression patterns were reproducible. For these six fusions, we observed reporter GUS expression only in early and late staged embryo sacs and not in leaves, stems, petals, and sepals. The GUS expression of the promoter∷GUS fusion with At1g26795, which encodes a self-incompatibility protein with a transmembrane domain, was detected strongly in dividing nuclei of the embryo sac from FG3 (Fig. 4E). We detected strong GUS expression in the embryo sac, including the egg cell and antipodal cells, from FG5 to FG7 (Fig. 5D). GUS expression of the promoter∷GUS fusion with At1g36340, which encodes a ubiquitin-conjugating enzyme, was detected in the closest nucleus to the chalazal end of the embryo sac at FG4 (Fig. 4F). In the late staged ovule, GUS activity was detected in only the antipodal cells (Fig. 5E) or was observed in the area of the degenerated antipodal cells at FG7. Both of the activities of the promoter∷GUS fusions with At2g20070, encoding a hypothetical protein, and At4g22050, encoding an aspartyl protease family, could be detected in FG1 of embryo sacs (Fig. 4, G and H). In particular, expression of the promoter∷GUS fusion with At2g20070 was detected in dividing nuclei of the embryo sac from FG1 to FG3 and in the chalazal end of the embryo sac at FG4. Expression of promoter∷GUS fusion with At4g22050 was detected in the chalazal end of the embryo sac from FG1. From FG5 to FG7, the activity of the promoter∷GUS fusion with At2g20070 or At4g22050 was detected in only the chalazal ends of the embryo sacs (Fig. 5, F and G). The expression of the promoter∷GUS fusion with At5g40260 was observed in the embryo sac from FG1 to FG7 (Figs. 4I and 5H). We could detect GUS expression of the promoter∷GUS fusion with At5g40260 in the functional megaspore at FG1 and in nuclei of the micropylar end of the embryo sac and the central vacuole from FG2 to FG4. In the late staged ovule, expression of the promoter∷GUS fusion with At5g40260 was detected strongly in the embryo sac. At5g40260 is annotated as encoding a protein of the nodulin MtN3 family expressed during root development of Medicago truncatula, but it also shows similarity with LIM7, a protein that is induced during the meiotic prophase in lily (Lilium longiflorum) microspores (Kobayashi et al., 1994). This gene was previously identified as stamen specific in Arabidopsis (Wellmer et al., 2004), and we have confirmed its expression in pollen (see below). The GUS activity of the promoter∷GUS fusion with At4g30590, encoding a plastocyanin-like domain containing protein, was weak but detectable in developing embryo sacs from FG4 to FG7 in a pattern similar to those of the promoter∷GUS fusions with At1g26795 or At5g40260 (data not shown). In the anthers, the activity of the promoter∷GUS fusion with At5g40260 showed strong staining beginning with floral stage 8, in which locules are visible in the stamen and microsporocytes are conspicuous, to floral stage 13, in which pollen grains are mature, indicating that this gene is expressed strongly in male gametophytes as well (for details, see Supplemental Fig. 2). In addition, GUS expression from the promoter∷GUS fusions with At1g26795, At1g36340, and At4g30590 could also be detected weakly in developing pollen from floral stages 9 to 13 (data not shown). Therefore, these four genes appear to be expressed in both male and female gametophytes.

Table III.

The genes used the promoter∷GUS fusion analysis

Genes were selected on the basis of the P value of <0.0050, fold change of >2.0, and signal intensity of >60.0 at wild-type siblings. Column headings and footnotes are the same as in Table II.

Affymetrix Codea	Gene IDb	Description	Early Staged Ovule				Late Staged Ovule
			WT1–3	spl1–3	FCc	P Valued	WTd1–d3	spl1–3, d1–d2	FCc	P Valued
261271_at	At1g26795	Self-incompatibility protein related	769	30	25.3	0.0000827	761	27	28.2	0.0000000
260124_at	At1g36340	Ubiquitin-conjugating enzyme, E2	584	38	15.3	0.0016607	467	34	13.8	0.0000023
265579_at	At2g20070	Hypothetical protein	661	48	13.8	0.0000412	487	36	13.4	0.0000000
254336_at	At4g22050	Aspartyl protease family protein	681	37	18.3	0.0000964	714	67	10.6	0.0000000
253634_at	At4g30590	Plastocyanin-like domain-containing protein	705	56	12.6	0.0000568	500	41	12.3	0.0000050
249401_at	At5g40260	Nodulin MtN3 family protein	566	37	15.2	0.0006028	427	30	14.3	0.0000038

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.

To examine the activity of the promoter∷GUS fusion with these six genes in sporophytic tissues, we stained 15-d-old seedlings for GUS expression (data summarized in Table IV). The GUS expression of At1g26795, At1g36340, At2g20070, and At4g30590 fusions was not detected in any part of the seedlings. Expression of the At5g40260 fusion could be detected in an occasional trichome, but as this expression was not consistent its significance is not clear. However, the expression of the promoter∷GUS fusion with At4g22050 was reproducibly detected in initiation sites of lateral roots and petioles, indicating that this gene is expressed sporophytically.

Table IV.

The summary for GUS expressions of six promoter∷GUS fusions in seedlings

Fifteen-day-old seedlings were used. To detect weak GUS activity, we used GUS staining solution without potassium ferricyanide/ferrocyanide.

Gene ID	GUS Expression
At1g26795	None
At1g36340	None
At2g20070	None
At4g22050	Lateral root, petiole
At4g30590	None
At5g40260	None except occasional trichome

To examine the functions in the embryo sac for these six genes, we analyzed the insertional mutants available from public collections listed in Supplemental Table II. However, we did not observe any embryo sac defects or other mutant phenotypes in these insertional mutants. To summarize, all six genes tested by promoter∷GUS fusions showed female gametophyte-specific expression within the developing ovules; four of the genes showed additional expression in the pollen, and one in sporophytic tissues.

DISCUSSION

Identification of Genes Expressed During Early and Late Developmental Stages of Embryo Sac Development

The female gametophyte in angiosperms is embedded in sporophyte tissue throughout development, making it technically challenging to isolate RNA from the developing gametophytes without extensive contamination from the surrounding sporophytic tissues. Here, we have isolated ovules from spl mutant homozygotes and phenotypically wild-type heterozygous siblings, and compared their expression profiles through the oligonucleotide array analysis. As spl ovules do not contain embryo sacs, genes with significantly higher transcripts in heterozygous sibling ovules than in spl ovules are presumptive embryo sac expressed genes. The RNA extracted from 2,500 to 3,000 ovules yielded enough RNA to perform each microarray experiment without any amplification. We avoided using amplified RNA to ensure comprehensive coverage of the transcriptome (Honys and Twell, 2004). The high correlation coefficient of >0.94 and Present call% of >70% validated good quality RNA used in our experiments.

In this study, two different ovule populations, corresponding to early and late developmental stages of the ovules, were used to perform the microarray assays. We used a set of three stringent criteria to narrow down the dataset to the most probable candidates for embryo sac-specific genes (i.e. <0.005 P value, a 2-fold cutoff, and >60 wild-type signal intensity). Using these criteria, we find 23 and 128 genes were identified in early and late staged ovules, respectively, and an additional 74 genes were expressed in both populations (Fig. 2; Table II). Thus, we identified more embryo sac genes expressed during later stages of megagametogenesis. This difference might arise from two sources. First, it could be due to the different complexities of the two ovule samples used in our study. While the embryo sacs of the early staged ovules undergo only three rounds of mitosis, the embryo sacs of the late staged ovules encompass more complex developmental processes, such as cellularization, nuclear fusion, and cell death, and the preparation during fertilization depends on cell-cell communication, such as pollen tube attraction and guidance and sperm nucleus recognition (Yang and Sundaresan, 2000; Hennig et al., 2004). Consistent with this idea, in a large-scale study of female gametophyte mutants, the majority of the identified genes are required after FG5 or during early embryogenesis, whereas only 16 of the identified genes are necessary before FG4 (Pagnussat et al., 2005). Second, the larger embryo sac of late staged ovules might have a more abundant pool of transcripts and this could facilitate the detection of more expressed genes in comparison with early staged ovules. This difference was even larger when we used a more stringent P value while maintaining the fold change and background signal criteria. For example, with a P value of <0.00001, 117 genes were identified for late staged ovules compared to only four genes for early staged ovules.

Comparison of Genes Identified in Female Gametophyte Development with Previous Sporophytic and Gametophytic Expression Studies

When we compared genes identified from our microarray analysis with genes previously shown to be involved in female gametophyte development through mutant analysis, we found very limited overlap. There may be two possible explanations for this. First, several genes identified through female gametophyte mutants, such as PRL (Springer et al., 1995), GFA2 (Christensen et al., 2002), and NOMEGA (Kwee and Sundaresan, 2003), also exhibit sporophytic expression. Genes with sporophytic expression in maternal ovule tissues were excluded from the gene set in the comparison of expression level of wild-type ovules and spl ovules. Consistent with their predicted sporophytic functions, genes required during megagametogenesis, such as PRL (At4g02060), GFA2 (At5g48030), and NOMEGA (At1g78770), were found to have high signal intensities for both wild-type and spl mutant ovules (Supplemental Table III).

Second, because of the high stringent selection criteria to minimize false positives in our microarray analysis, even genes specifically involved in female gametophyte development and function could be eliminated in our analysis if they do not have high expression in the embryo sacs. For example, the genes of the FIE-FIS-MEA Polycomb Group complex are not present in the set of embryo sac genes identified in this study. It is known that MEA and FIS2 are expressed in polar nuclei and central cell by promoter∷GUS fusion analysis (Ohad et al., 1999; Vielle-Calzada et al., 1999; Luo et al., 2000). As mentioned above, we applied stringent criteria for the microarray analysis to minimize the number of false positives. MEA (FIS1, At1g02580) and FIS2 (At2g35670) showed higher expression in wild type versus spl mutants in the late staged ovules, but they did not satisfy some criteria, such as fold change of >2.0 in the case of MEA or higher signal intensity than the background signal of the Affymetrix control genes in the case of FIS2 (Supplemental Table III). In the case of FIE (FIS3, At3g20740), because FIE is expressed also in sporophyte tissues (Luo et al., 2000), it is eliminated in our analysis from the final dataset of female gametophyte genes (Supplemental Table III).

When the embryo sac genes were compared with female gametophyte genes obtained through the study using Ds tagging mutants in Arabidopsis (Pagnussat et al., 2005), expressed sequence tag analysis of maize (Zea mays) embryo sac (http://www.pgec.usda.gov/McCormick/McCormick/ResearchTopics/Gametes/Gametesindex.htm), and with carpel-specific genes by transcriptome comparison of floral homeotic mutants (Wellmer et al., 2004), 28 genes matched with our identified genes (Supplemental Table IV). The comparison with our female gametophyte mutant study (Pagnussat et al., 2005) showed that gene At2g34130 corresponded to MEE19, which resulted in maternal effect-arrested zygote and endosperm development, and gene At5g44700 corresponded to EDA23, which resulted in varying arrested stages of embryo sac development. The failure to identify more genes corresponding to the female gametophyte mutants arises from both of the factors discussed above, i.e. sporophytic expression of the gene in maternal ovule tissues and/or very low expression of the gene in the gametophyte.

The genes identified in this study are predicted to be either up-regulated in female gametophytes, as compared to the maternal ovule tissue, or specific for the female gametophyte. The higher fraction of unknown and hypothetical proteins for the genes identified here (33%), as compared to the fraction in the whole genome (21%), suggests that many genes of currently unknown function might have specific functions in the gametophytes. We compared the 225 genes identified here with transcriptome data of sporophytic tissues, using ATH1 Genome Array datasets for seedlings at open cotyledon stage (stage 0.7), leaves (stage 6.0), petiole (stage 3.9), stems (stage 6.1), roots, and root hair zone (stage 1.02) provided by the Nottingham Arabidopsis Stock Centre's microarray database (http://affymetrix.arabidopsis.info/narrays/experimentbrowse.pl) and reanalyzed by Honys and Twell (2004). As a result, 101 genes (44.9%) of the 225 identified genes were found to have no transcriptome data in sporophytic tissues and appear to be gametophyte-specific genes. Further comparison of the 101 putative gametophyte-specific genes with data from expression studies that contain male gametophytes showed that 17 genes were identified as pollen-specific genes (Honys and Twell, 2004) and nine genes were analyzed as stamen-specific genes representing potential male gametophyte genes (Wellmer et al., 2004). Therefore, of the 101 genes that are not expressed in sporophytic tissues, 19 genes might be transcribed in both gametophytes and 82 genes might be specific to the female gametophyte (Supplemental Table IV). These conclusions are reflected in the results from the promoter∷GUS fusions examined. Out of six promoters that exhibited embryo sac-specific expression within the ovule, five did not show detectable expression in the sporophytic tissues examined, and four (At1g26795, At1g36340, At4g30590, and At5g40260) showed expression in the pollen. Examination of the insertional mutants revealed that none of the six mutant genes resulted in an observable phenotype in the embryo sac. This absence of mutant phenotypes could be due to redundancy. For example, At2g23990, which is the closest relative of At4g30590, was also detected as expressed in embryo sacs in our array analysis (Table II) and therefore might be playing the same role as At4g30590 in the same pathways during embryo sac development. Another of the genes examined, At4g22050, is closely related to two other genes (At1g62230 and At4g04460) of the aspartyl protease family. The gene At5g40260, which we found to be strongly expressed in embryo sacs and pollen, is related to four other Arabidopsis genes (At3g16690, At4g15920, At1g66770, and At5g62850) annotated as members of the nodulin MtN3 family. Similar outcomes were recently obtained by Nawy et al. (2005) in their analysis of genes expressed in the Arabidopsis root quiescent center; they also found that disruptions of identified genes with specific expression in the quiescent center did not yield observable mutant phenotypes, again possibly reflecting the extent of functional redundancy in the Arabidopsis genome.

CONCLUSION

Our study uses a comparative expression profiling strategy to provide a window into the female gametophyte transcriptome in Arabidopsis and to identify a subset of genes comprising this transcriptome. Nearly one-half of the identified genes have not previously been identified in expression profiling of sporophytic tissues, and there is only a partial overlap with genes identified from the expression profile of pollen. Mutational analysis of these genes will be important to undertake in future studies to understand their precise roles in embryo sac development and function. The low-level signals for many genes, due to the limiting amounts of plant material as well as low expression levels, suggest that a much larger number of genes may be found to be specific for the female gametophyte that are excluded from the list of genes presented here by the stringent criteria used to compile the final gene set. However, the complete unfiltered dataset that has been generated, which is provided as supplemental material and is also available from the laboratory Web site online (http://sundarlab.ucdavis.edu/), can be used to examine the possibility of female gametophyte expression for any specific gene in the Arabidopsis genome. The biological functions of most Arabidopsis genes remain to be determined, and the expression data in this study can assist in identifying putative functions in the gametophyte that would be missed in studies of sporophytic expression.

MATERIALS AND METHODS

Plant Material and Plant Growth Condition

Seeds of Landsberg erecta were directly or after spreading on Murashige and Skoog plates transferred to soil with 16-h-light/8-h-dark cycle at 22°C with 60% humidity. After the spl mutants were selected on Murashige and Skoog kanamycin plates, they were grown on the soil under the condition as described previously (Yang et al., 1999). The maintenance of the spl line and segregation analysis were carried out according to Sundaresan et al. (1995).

Genotyping and Ovule Collection of spl/spl and spl/SPL Plants

To confirm the spl homozygous or heterozygous plants, genotyping was performed using SPL primers SPL-F (T5′-GGCGAGATCCGGACAGAGAC-3′) and SPL-R (5′-AGAAGCGTTAAACATTTGAGGATT-3′) and Ds primers DS 3-3A (T5′-TCGTTTCCGTCCCGCAAGT-3′) or DS 5 to 3A (5′-CGGTCGGTACGGGATTTTCC-3′). PCR was performed in a total volume of 10 μL containing 1× PCR buffer (Fisher Scientific) with 1.5 mm MgCl₂, 0.2 mm of each dNTP, 5 pmol of each primer, and 2 units of Taq DNA polymerase for 34 cycles at an annealing temperature of 55°C.

The ovule samples were dissected from placenta of ovaries with needles and collected under dry ice on the basis of the floral stage. The collected ovules were randomly cleared overnight with Hoyer's solution (Liu and Meinke, 1998) to confirm the embryo sac stages. The cleared whole-mount preparations and observation were performed as described by Pagnussat et al. (2005).

RNA Extraction, Probe Preparation, and Array Hybridization

Total RNA was extracted from ovule samples collected on the basis of the developmental stages using the RNeasy Plant Mini kit (Qiagen) according to the manufacturer's protocols. The yield and RNA purity were determined by the ratio of absorbencies at 260 nm/280 nm wavelengths. RNA integrity was checked by running 1 μL of every RNA preparation on the glyoxylated RNA gel (Sambrook and Russell, 2001). The first- and second-strand cDNAs were synthesized from 5 μg total RNA using the MessageAmp RNA kit (Ambion), which is based on the manual of Dr. James Everwine (Van Gelder et al., 1990). Biotin-labeled target cRNAs were prepared by cDNA in vitro transcription using the BioArray High-Yield RNA transcript labeling kit (Enzo Biochem) and cleaned using GeneChip Sample Cleanup Module (Affymetrix). The 15 μg labeled target cRNAs were fragmented and hybridized with Arabidopsis ATH1 Genome Arrays for 16 h at 45°C as described in the Affymetrix GeneChip expression analysis technical manual. The hybridizations and scanning were carried out at the University of California Davis Microarray Facility.

Data Acquisition and Statistical Analysis

Raw data were processed with Affymetrix Microarray Suite 5.0. The raw data were converted into numbers by calculations of the PM-only model (Li and Wong, 2001a) using dChip program version 1.3 (http://www.dchip.org/). Pearson's correlation coefficient (r) was calculated to examine the relation between microarrays using package R program version 1.6.1 (Ihaka and Gentleman, 1996). Microsoft Excel (Microsoft) was used in the calculation of P value by Student's t test and management of the microarray data.

Plant Transformation

To construct the promoter∷GUS cassettes for six genes (At1g26795, At1g36340, At2g20070, At4g22050, At4g30590, and At5g40260), promoter fragments were amplified by PCR using primers (Supplemental Table V) and inserted into EcoRI and NcoI sites upstream of the GUS gene (Jefferson, 1987) of pRITA. The NotI fragments from these plasmids were subcloned into pMLBART for transferring into plants. For plant transformation, the vectors were transferred into Agrobacterium tumefaciens strain ASE (Fraley et al., 1985). Transformations were performed on wild-type Landsberg erecta or Columbia-0 by floral dip procedures (Clough and Bent, 1998). The seeds obtained from the T0 promoter∷GUS transformants were selected by spraying 0.1% Basta, and Basta-resistant plants of the T1 generation were analyzed for the presence of the transgene by PCR using the primers of Basta-resistance gene, BA-F (5′-CCGTACCGAGCCGCAGGAAC-3′) and BA-R (5′-CAGATCTCGGTGACGGGCAGGAC-3′), and were self-crossed to collect seeds of T2 generation.

GUS Assays and Image Processing

For the expression analysis of GUS fused with promoter, the seeds of the T2 generation from each transformant were germinated on soil without sterilization, and then they were selected with 0.1% Basta. Inflorescences from soil-grown plants were transferred to microtiter well containing 1 mL of GUS staining solution (50 mm sodium phosphate buffer, pH 7.0, 10 mm EDTA, 0.1% Triton X-100, 2 mm potassium ferricyanide, 2 mm potassium ferrocyanide, 1 mg/mL X-Gluc). The microtiter dish was placed under vacuum for 10 min in desiccators. After release of the vacuum, the dish was covered with aluminum foil and incubated at 37°C overnight (Sundaresan et al., 1995). The solution was removed, and the tissues were cleared in 0.85% sodium chloride/70% ethanol at room temperature. For detection of weak GUS expression, GUS staining was performed under the absence of potassium ferricyanide and ferrocyanide, too. To see clear and specific GUS expression, the potassium ferricyanide and ferrocyanide concentrations were readjusted in the range of 1.3 to approximately 10 mm. The pistils were dissected with needles and cleared in Hoyer's solution (Liu and Meinke, 1998), and the cleared ovules were observed under a Zeiss Axioplan imaging 2 microscope under differential interference contrast optics. Images were captured on an Axiocam HRC CCD camera (Zeiss) using the Axiovision program (version 3.1). All images were processed for publication using Adobe Photoshop CS (Adobe Systems).