PMC

Genes essential for female gametogenesis, fertilization, and seed development are present in the embryo sac transcriptome datasets. (a) Chromosomal locations of 35 essential genes. Five genes that are described in the current work are shown in blue. Description of the mutants and corresponding references are given in Additional data file 5. (b) Five genes and the locations of corresponding mutant alleles described in this work. Exons are shaded in orange. The genes were named after the following Goddesses: KERRIDWIN, the Welsh triple Goddess of trinity known for nurturing children; OMISHA, Indian Goddess of birth and death; FREYA, the Norse Goddess of fertility; and ILITHYIA, the Greek Goddess of childbirth. HOG1, HOMOLOGY DEPENDENT GENE SILENCING 1; LB and RB, left and right borders of the T-DNA. (c) Mutants were identified based on infertile ovules (ken-1) or seed abortion (hog1-6, oma-1, fey-1, and ila-1). The arrows identify the defective ovules. Scale bar: 100 μm in panel c.

Mutants arrested late in seed development. (a) Shown is a scheme of seed development in Arabidopsis. A globular embryo (EM) develops into heart stage (1). Note that the peripheral endosperm nuclei surrounding the globular embryo are organized into three distinct domains: micropylar endosperm (ME), chalazal endosperm (CE), and free nuclear endosperm (FNE). Following rapid cellularization of endosperm, a torpedo stage embryo and then an upturned-U stage embryo is formed (2). (b-e) Morphology of wild-type seed development corresponding to representative events described above. (f) In oma-1 the embryo arrested at the mid-globular stage. The size of cells in embryo and endosperm were larger than that in (g) the wild type. (h,i) In fey-1 the embryo arrested at around the late globular stage. Note that the cells of the embryo and suspensor were large, and the suspensor displays a bend due to the irregularly bulged cells (panel i, arrow). (j) The majority of the ila-1 embryos arrested when they were at upturned U stage. (k) A small fraction of late-heart ila-1 embryos could also be observed. Scale bars: 10 μm for panels b, f, h, j, and k; and 20 μm for panels c, d, e, g, and i.

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 μm in panels b and c.

Confirmation of embryo sac expression for selected genes. Embryo sac expression of nine candidate genes is shown by in situ hybridization (panels a, c, d, f, g, and i) or histochemical reporter gene (GUS) analysis (b, e, and h). Illustrated is the in situ expression of broadly expressed genes: (a) AT1G78940 (encoding a protein kinase that is involved in regulation of cell cycle progression), (c) AT5G40260 (encoding a nodulin), and (d) AT4G30590 (encoding a plastocyanin). Also shown is the restricted expression of (f) AT3G61740 (encoding the trithorax-like protein ATX3), (g) AT5G50915 (encoding a TCP transcription factor), and (i) AT5G60270 (encoding a protein kinase). The corresponding sense control for panels a, b, c, d, f, g, and i did not show any detectable signal (data not shown). GUS staining: (b) an enhancer-trap line for AT4G01970 (encoding a galactosyltransferase) shows embryo sac expression, (e) a promoter-GUS line for AT1G80370 (encoding CYCLIN A2;4) shows a strong and specific expression in the embryo sac and endothelium (insert: shows several ovules at lower magnification), and (h) a promoter-GUS line for AT1G28220 (encoding the purine permease PUP3) shows synergid specific expression (insert; note the pollen-specific expression of PUP3-GUS when used as a pollen donor on a wild-type pistil). CC, central cell; EC, egg cell; SC, synergids. Scale bars: 50 μm in panels a to i; and 100 μm and 50 μm in the inserts of panels e and h, respectively.

Gain of expression in the sporophyte in the absence of a functional embryo sac: expression analysis in the coatlique (coa) mutant. (a) RT-PCR for 11 genes in coa and wild-type (WT) pistils. Equal loading of both coa and WT cDNA templates in PCR was monitored by expression of ACT11. SUP, SUPERMAN. Also shown are in situ expression patterns of the following genes in coa pistil tissues: (b) AT4G12410, encoding an auxin-responsive Small Auxin Up RNA (SAUR) protein; (c) AT1G75580, encoding an auxin-responsive protein; (d) AT5G03200, encoding a C3HC4-type RING finger protein; and (e) at5g15980, encoding a PPR repeat containing protein. The corresponding sense control probes did not show any expression (data not shown). (f) AT4G12410 did not show any detectable expression pattern in wild-type pistils. The other four genes exhibited spatial expression patterns in the wild-type ovule and carpel tissues comparable to that of coa, but their wild-type expression levels were much lower than in coa (data not shown). (g) We initially identified the over-expression of STM in the ovule tissues of spl (sensu microarray data), and confirmed that this gene is over-expressed in the carpel and ovules of coa as well (panels a and g). (h) A comparable but less intense spatial expression pattern of STM was seen in wild-type pistils. Scale bars: 100 μm in panels b to h.

Female gametophytic and early zygotic mutant phenotypes highlight the essential role of corresponding genes for reproductive development. (a) A cartoon showing the ontogeny of the wild-type female gametophyte in Arabidopsis and the early transition to seed development. A haploid functional megaspore (FM) develops from a diploid megaspore mother cell (MMC) upon two meiotic divisions (1). Three syncitial mitotic divisions (2) convert the FM into an eight-nuclear cell. Upon nuclear migration, cellularization, nuclear fusion and differentiation (3), a cellularized seven-celled embryo sac forms. It contains an egg cell (EC) and two synergid cells (SC) at the micropylar pole, three antipodals (AP) at the chalazal pole, and one vacuolated homo-diploid central cell (CC) in the middle. Subsequently, the AP cells degenerate. Degeneration of one SC precedes the entry of one pollen tube (PT), and two sperm cells (SP) independently fertilize the egg and central cell, leading to the development of a diploid embryo (EM) and triploid endosperm (EN) respectively. SUS, suspensor, VN, vegetative nucleus. (b-f) Morphology of wild-type ovules corresponding to representative events described above is depicted (ii indicates inner integuments, and oi indicates outer integuments). Both synchronous and asynchronous free nuclear mitotic divisions (as shown in panel e; arrows) lead to development of the free nuclear endosperm (FNE) as shown in panel f. The insert in panel e depicts a developing zygote (ZY). (g) In kerridwin (ken-1), two polar nuclei in the central cell fail to fuse. (h) Female gametophyte development did not initiate beyond the one-nucleate embryo sac stage (arrows) in frigg (fig-1). (i-l) Anomalies in early endosperm and zygotic development in hog1 (homology dependent gene silencing 1) mutants. The zygote did not develop beyond single cell stage, and subsequent divisions and cytokinesis did not occur (panel i, j, and k). The arrows in panels i and j identify the irregular nature of free nuclear mitotic divisions in hog-1 endosperm. The endosperm nuclei were irregular in size and they were often clustered. Compare the large and small irregular endosperm nuclei in hog1-6 (panel l) with the regular free nuclear endosperm nuclei in (m) the wild type. Scale bars: 20 μm for panels d to k, and the insert of panel e; and 50 μm in panels b, c, l, and m.