In flowering plants, signaling between the male pollen tube and the synergid cells of the female gametophyte is required for fertilization. In the Arabidopsis thaliana mutant feronia (fer), fertilization is impaired; the pollen tube fails to arrest and thus continues to grow inside the female gametophyte. FER encodes a synergid-expressed, plasma membrane-localized receptor-like kinase. We found that the FER protein accumulates asymmetrically in the synergid membrane at the filiform apparatus. Interspecific crosses using pollen from Arabidopsis lyrata and Cardamine flexuosa on A. thaliana stigmas resulted in a fer-like phenotype that correlates with sequence divergence in the extracellular domain of FER. Our findings show that the female control of pollen tube reception is based on a FER-dependent signaling pathway, which may play a role in reproductive isolation barriers.

The development of multicellular organisms is controlled by differential gene expression whereby cells adopt distinct fates. A spatially resolved view of gene expression allows the elucidation of transcriptional networks that are linked to cellular identity and function. The haploid female gametophyte of flowering plants is a highly reduced organism: at maturity, it often consists of as few as three cell types derived from a common precursor [1, 2]. However, because of its inaccessibility and small size, we know little about the molecular basis of cell specification and differentiation in the female gametophyte. Here we report expression profiles of all cell types in the mature Arabidopsis female gametophyte. Differentially expressed posttranscriptional regulatory modules and metabolic pathways characterize the distinct cell types. Several transcription factor families are overrepresented in the female gametophyte in comparison to other plant tissues, e.g., type I MADS domain, RWP-RK, and reproductive meristem transcription factors. PAZ/Piwi-domain encoding genes are upregulated in the egg, indicating a role of epigenetic regulation through small RNA pathways-a feature paralleled in the germline of animals [3]. A comparison of human and Arabidopsis egg cells for enrichment of functional groups identified several similarities that may represent a consequence of coevolution or ancestral gametic features.

In plants, members of microRNA (miRNA) families are often predicted to target the same or overlapping sets of genes. It has thus been hypothesized that these miRNAs may act in a functionally redundant manner. This hypothesis is tested here by studying the effects of elimination of all three members of the MIR164 family from Arabidopsis. It was found that a loss of miR164 activity leads to a severe disruption of shoot development, in contrast to the effect of mutation in any single MIR164 gene. This indicates that these miRNAs are indeed functionally redundant. Differences in the expression patterns of the individual MIR164 genes imply, however, that redundancy among them is not complete, and that these miRNAs show functional specialization. Furthermore, the results of molecular and genetic analyses of miR164-mediated target regulation indicate that miR164 miRNAs function to control the transcript levels, as well as the expression patterns, of their targets, suggesting that they might contribute to developmental robustness. For two of the miR164 targets, namely CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, we provide evidence for their involvement in the regulation of growth and show that their derepression in miR164 loss-of-function mutants is likely to account for most of the mutant phenotype.

Double fertilization is the defining characteristic of flowering plants. However, the molecular mechanisms regulating the fusion of one sperm with the egg and the second sperm with the central cell are largely unknown. We show that gamete interactions in Arabidopsis depend on small cysteine-rich EC1 (EGG CELL 1) proteins accumulating in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 (HAPLESS 2/GENERATIVE CELL SPECIFIC 1) to the cell surface. Furthermore, fertilization studies with ec1 quintuple mutants show that successful male-female gamete interactions are necessary to prevent multiple-sperm cell delivery. Our findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions.

Ovules of higher plants are the precursors of seeds. Ovules emerge from placental tissue inside the gynoecium of flowers. Three elements, funiculus, chalaza, and nucellus, can be distinguished along the proximal-distal axis of the outgrowing radially symmetrical ovule primordium. The asymmetric initiation of the outer integument marks the switch to adaxial-abaxial development, which leads to the formation of a bilaterally symmetrical ovule. The putative transcriptional regulator NOZZLE (NZZ) plays a role in mediating this transition by controlling the timing of expression of the putative transcriptional regulator INNER NO OUTER (INO) in an abaxial domain of the chalaza, from where the outer integument initiates. Integument formation depends on the homeobox gene WUSCHEL (WUS), which is expressed in the nucellus and is sufficient to induce integuments non-cell autonomously from a region adjacent to its expression domain. In this study, we describe the expression pattern of the homeobox-leucine zipper gene PHABULOSA (PHB) during ovule development, demonstrating that adaxial-abaxial polarity is established from the very beginning of ovule development. Furthermore, we examined the expression pattern of PHB, INO, and WUS in ovules of plants, which are affected in integument initiation and thus defective in the transition from proximal-distal to adaxial-abaxial development. We found that NZZ is required to restrict PHB expression to the distal chalaza, from where the inner integument initiates. PHB expression is not established in the distal chalaza of two mutants, aintegumenta (ant) and wus, which fail to form integuments. Furthermore, we suggest that one mechanism by which WUS controls integument formation is by establishing the chalaza and that outer and inner integument identity determination depends on additional region-specific factors. In addition, we present evidence that NZZ is essential for the normal nucellar expression pattern of WUS. Thus, both WUS and PHB affect processes downstream of NZZ action during the transition from proximal--distal to adaxial--abaxial ovule development.

Expression profiling genes essential for plant reproduction

Genetic subtraction and expression profiling of wild-type Arabidopsis and a sporophytic mutant lacking an embryo sac identified 1,260 genes expressed in the embryo sac; a total of 527 genes were identified for their expression in ovules of mutants lacking an embryo sac.

SUMMARYIn contrast to animals, the life cycle of higher plants alternates between a gamete-producing (gametophyte) and a spore-producing (sporophyte) generation. The female gametophyte of angiosperms consists of four distinct cell types, including two gametes, the egg and the central cell, which give rise to embryo and endosperm, respectively. Based on a combined subtractive hybridization and virtual subtraction approach in wheat (Triticum aestivum L.), we have isolated a class of transcription factors not found in animal genomes, the RKD (RWP-RK domain-containing) factors, which share a highly conserved RWP-RK domain. Single-cell RT-PCR revealed that the genes TaRKD1 and TaRKD2 are preferentially expressed in the egg cell of wheat. The Arabidopsis genome contains five RKD genes, at least two of them, AtRKD1 and AtRKD2, are preferentially expressed in the egg cell of Arabidopsis. Ectopic expression of the AtRKD1 and AtRKD2 genes induces cell proliferation and the expression of an egg cell marker. Analyses of RKD-induced proliferating cells exhibit a shift of gene expression towards an egg cell-like transcriptome. Promoters of selected RKD-induced genes were shown to be predominantly active in the egg cell and can be activated by RKD in a transient protoplast expression assay. The data show that egg cell-specific RKD factors control a transcriptional program, which is characteristic for plant egg cells.

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.