The female reproductive unit of flowering plants, the haploid female gametophyte, is highly reduced relative to other land plants. We show that patterning of the Arabidopsis female gametophyte depends on an asymmetric distribution of the hormone auxin during its syncitial development. Furthermore, this auxin gradient is correlated with location-specific auxin biosynthesis, rather than auxin efflux that directs patterning in the diploid sporophytic tissues comprising the rest of the plant. Manipulation of auxin responses or synthesis induces switching of gametic and nongametic cell identities and specialized nonreproductive cells to exhibit attributes presumptively lost during angiosperm evolution. These findings may account for the unique egg cell specification characteristic of angiosperms and the formation of seeds with single diploid embryos while containing endosperm that can have variable numbers of parental haploid genomes.

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development.

SUMMARYGEX1 is a plasma membrane protein that is conserved among plant species, and has previously been shown to be expressed in sperm cells and some sporophytic tissues. Here we show that GEX1 is also expressed in the embryo sac before cellularization, in the egg cell after cellularization, in the zygote/embryo immediately after fertilization and in the pollen vegetative cell. We functionally characterize GEX1 in Arabidopsis thaliana, and show that it is a versatile protein that performs functions during male and female gametophyte development, and during early embryogenesis. gex1-1/+ plants, which synthesize a truncated GEX1 mRNA encoding a protein lacking the predicted cytoplasmic domain, but still targeted to the plasma membrane, had embryos that arrested before the pre-globular stage. gex1-3/+ plants, carrying a null GEX1 allele, had defects during male and female gametophyte development, and during early embryogenesis. Using an antisense GEX1 transgenic line we demonstrate that the predicted GEX1 extracellular domain is sufficient and necessary for GEX1 function during the development of both gametophytes. The predicted cytoplasmic domain is necessary for correct early embryogenesis and mediates homodimer formation at the plasma membrane. We propose that dimerization of GEX1 in the zygote might be an upstream step in a signaling cascade regulating early embryogenesis.

ARR22 (At3g04280) is a novel Type A response regulator whose function in Arabidopsis is unknown. RT-PCR analysis has shown that expression of the gene takes place in flowers and developing pods with the tissues accumulating different proportions of splice variants. Spatial analysis of expression, using ARR22::GUS plants as a marker, has revealed that the reporter protein accumulates specifically at the junction between the funiculus and the chalazal tissue. Expression can be up-regulated at this location by wounding the developing seed. A detailed analysis has failed to detect ARR22 expression at any other sites and, to support this assertion, the only evidence for tissue ablation in ARR22::Barnase plants is during seed development, with the consequence that embryo growth is attenuated. Ectopic expression of ARR22, driven by either the CaMV 35S or the pea plastocyanin (PPC) promoters, resulted in the generation of plants exhibiting extremely stunted root and shoot growth. No viable progeny could be isolated from the PPC::ARR22 transgenic lines. An RT-PCR analysis of a recently annotated gene (ARR24-At5g26594), that exhibits 66% amino acid similarity to ARR22, has shown that expression is also predominantly in floral and silique tissues. Examination of ARR24::GUS plants has revealed that the activity of the promoter is primarily restricted to pollen grains indicating that this gene is unlikely to display an overlapping function with ARR22. Analyses of individual KO lines of either ARR22 or ARR24 have failed to identify a mutant phenotype under the growth conditions employed and the double knockout ARR22/ARR24 line is also indistinguishable from wild-type plants. These results are discussed in the light of the proposed role of response regulators in plant growth and development.

SummaryPlant reproduction involves gamete production by a haploid generation, the gametophyte. For flowering plants, a defining characteristic in the evolution from the 'naked-seed' plants, or gymnosperms, is a reduced female gametophyte, comprising just seven cells of four different types -a microcosm of pattern formation and gamete specification about which only little is known. However, several genes involved in the differentiation, fertilization and post-fertilization functions of the female gametophyte have been identified and, recently, the morphogenic activity of the plant hormone auxin has been found to mediate patterning and egg cell specification. This article reviews recent progress in understanding the pattern formation, maternal effects and evolution of this essential unit of plant reproduction. Key words: Embryo sac, Gametophyte, Flowering plant, Reproduction, Gametes IntroductionThe life cycle of land plants involves an alternation of generations between a haploid gametophyte (see Glossary, Box 1) and a diploid sporophyte (see Glossary, Box 1). Whereas animal gametes are formed directly after meiosis, plant gametes are produced only after growth of the multicellular haploid gametophyte. The morphological complexity of the haploid generation ranges from the macroscopic moss gametophytes, which dwarf the sporophyte, to the three-celled male gametophyte (pollen) and seven-celled female gametophyte (embryo sac) that are characteristic of most flowering plants (Maheshwari, 1950). The latter evolved through an extreme reduction from the female gametophytes of the gymnosperms (see Glossary, Box 1), which frequently contain over a thousand cells, and is considered a key innovation in the evolution of flowering plants (reviewed by Friedman and Williams, 2003). These 'stripped down to essentials' female gametophytes confer two major defining characteristics of the flowering plants. First, they are small enough to be packaged within an ovary. Second, they generate two gametes that undergo double-fertilization to produce the nutritive tissue called endosperm concordantly with the embryo, which allows more efficient resource allocation to fertilized seeds (reviewed by Raghavan, 2003). The reduced female gametophyte of flowering plants enabled much more rapid seed setting (i.e. the production of seeds during reproductive growth) than is possible in gymnosperms, allowing for habitat adaptations that require short reproductive cycles and facilitating the expansion of flowering plants into diverse ecological niches.Despite the crucial importance of the female gametophyte of flowering plants, much remains to be learnt about its development and overall biology, partly because it is a highly inaccessible structure. The past few years, however, have seen some exciting progress in the field. Here, we focus primarily on the female gametophyte of the model plant Arabidopsis, which has been studied more extensively than that of other plants, and review recent advances in the understanding of the patterning, the maternal e...