The life cycle of flowering plants alternates between two heteromorphic generations: a diploid sporophytic generation and a haploid gametophytic generation. During the development of the plant reproductive lineages – the germlines – typically, single sporophytic (somatic) cells in the flower become committed to undergo meiosis. The resulting spores subsequently develop into highly polarized and differentiated haploid gametophytes that harbour the gametes. Recent studies have provided insights into the genetic basis and regulatory programs underlying cell specification and the acquisition of reproductive fate during both sexual reproduction and asexual (apomictic) reproduction. As we review here, these recent advances emphasize the importance of transcriptional, translational and post-transcriptional regulation, and the role of epigenetic regulatory pathways and hormonal activity.

The plant life cycle alternates between a diploid sporophytic and a haploid gametophytic generation. The female gametophyte (FG) of flowering plants is typically formed through three syncytial mitoses, followed by cellularisation that forms seven cells belonging to four cell types. The specification of cell fates in the FG has been suggested to depend on positional information provided by an intrinsic auxin concentration gradient. The goal of this study was to develop mathematical models that explain the formation of this gradient in a syncytium. Two factors were proposed to contribute to the maintenance of the auxin gradient in Arabidopsis FGs: polar influx at early stages and localised auxin synthesis at later stages. However, no gradient could be generated using classical, one-dimensional theoretical models under these assumptions. Thus, we tested other hypotheses, including spatial confinement by the large central vacuole, background efflux and localised degradation, and investigated the robustness of cell specification under different parameters and assumptions. None of the models led to the generation of an auxin gradient that was steep enough to allow sufficiently robust patterning. This led us to re-examine the response to an auxin gradient in developing FGs using various auxin reporters, including a novel degron-based reporter system. In agreement with the predictions of our models, auxin responses were not detectable within the FG of Arabidopsis or maize, suggesting that the effects of manipulating auxin production and response on cell fate determination might be indirect.

The directional growth of the pollen tube from the stigma to the embryo sac in the ovules is regulated by pollen-pistil interactions based on intercellular communication. Although pollen tube growth is regulated by the cytoplasmic Ca 2+ concentration ([Ca 2+ ] cyt ), it is not known whether [Ca 2+ ] cyt is involved in pollen tube guidance and reception. Using Arabidopsis expressing the GFP-based Ca 2+ -sensor yellow cameleon 3.60 (YC3.60) in pollen tubes and synergid cells, we monitored Ca 2+ dynamics in these cells during pollen tube guidance and reception under semi-in vivo fertilization conditions. In the pollen tube growing towards the micropyle, pollen tubes initiated turning within 150 μm of the micropylar opening; the [Ca 2+ ] cyt in these pollen tube tips was higher than in those not growing towards an ovule in assays with myb98 mutant ovules, in which pollen tube guidance is disrupted. These results suggest that attractants secreted from the ovules affect Ca 2+ dynamics in the pollen tube. [Ca 2+ ] cyt in synergid cells did not change when the pollen tube grew towards the micropyle or entered the ovule. Upon pollen tube arrival at the synergid cell, however, [Ca 2+ ] cyt oscillation began at the micropylar pole of the synergid, spreading towards the chalazal pole. Finally, [Ca 2+ ] cyt in the synergid cell reached a maximum at pollen tube rupture. These results suggest that signals from the pollen tube induce Ca 2+ oscillations in synergid cells, and that this Ca 2+ oscillation is involved in the interaction between the pollen tube and synergid cell.

In plants, gametes, along with accessory cells, are formed by the haploid gametophytes through a series of mitotic divisions, cell specification and differentiation events. How the cells in the female gametophyte of flowering plants differentiate into gametes (the egg and central cell) and accessory cells remains largely unknown. In a screen for mutations that affect egg cell differentiation in Arabidopsis , we identified the wyrd ( wyr ) mutant, which produces additional egg cells at the expense of the accessory synergids. WYR not only restricts gametic fate in the egg apparatus, but is also necessary for central cell differentiation. In addition, wyr mutants impair mitotic divisions in the male gametophyte and endosperm, and have a parental effect on embryo cytokinesis, consistent with a function of WYR in cell cycle regulation. WYR is upregulated in gametic cells and encodes a putative plant ortholog of the inner centromere protein (INCENP), which is implicated in the control of chromosome segregation and cytokinesis in yeast and animals. Our data reveal a novel developmental function of the conserved cell cycle-associated INCENP protein in plant reproduction, in particular in the regulation of egg and central cell fate and differentiation.

The precise delivery of male to female gametes during reproduction in eukaryotes requires complex signal exchanges and a flawless communication between male and female tissues. In angiosperms, molecular mechanisms have recently been revealed that are crucial for the dialog between male (pollen tube) and female gametophytes required for successful sperm delivery. When pollen tubes reach the female gametophyte, they arrest growth, burst and discharge their sperm cells. These processes are under the control of the female gametophyte via the receptor-like serine-threonine kinase (RLK) FERONIA(FER). However, the male signaling components that control the sperm delivery remain elusive. Here, we show that ANXUR1 and ANXUR2 ( ANX1, ANX2 ), which encode the closest homologs of the FER-RLK in Arabidopsis , are preferentially expressed in pollen. Moreover,ANX1-YFP and ANX2-YFP fusion proteins display polar localization to the plasma membrane at the tip of the pollen tube. Finally, genetic analyses demonstrate that ANX1 and ANX2 function redundantly to control the timing of pollen tube discharge as anx1 anx2 double-mutant pollen tubes cease their growth and burst in vitro and fail to reach the female gametophytes in vivo. We propose that ANX-RLKs constitutively inhibit pollen tube rupture and sperm discharge at the tip of growing pollen tubes to sustain their growth within maternal tissues until they reach the female gametophytes. Upon arrival, the female FER -dependent signaling cascade is activated to mediate pollen tube reception and fertilization, while male ANX -dependent signaling is deactivated, enabling the pollen tube to rupture and deliver its sperm cells to effect fertilization.

Early seed development of sexually reproducing plants requires both maternal and paternal genomes but is prominently maternally influenced. A novel gametophytic maternal-effect mutant defective in early embryo and endosperm development, glauce ( glc ), has been isolated from a population of Arabidopsis Ds transposon insertion lines. The glc mutation results from a deletion at the Ds insertion site, and the molecular identity of GLC is not known. glc embryos can develop up to the globular stage in the absence of endosperm and glc central cells appear to be unfertilized. glc suppresses autonomous endosperm development observed in the fertilization-independent seed ( fis ) class mutants. glc is also epistatic to mea , one of the fis class mutants, in fertilized seeds, and is essential for the biparental embryonic expression of PHE1 , a repressed downstream target of MEA . In addition, maternal GLC function is required for the paternal embryonic expression of the ribosome protein gene RPS5a and the AMP deaminase gene FAC1 ,both of which are essential for early embryo and endosperm development. These results indicate that factors derived from the female gametophyte activate a subset of the paternal genome of fertilized seeds.

In flowering plants, male fertility depends on proper cell differentiation in the anther. However, relatively little is known about the genes that regulate anther cell differentiation and function. Here, we report the analysis of a new Arabidopsis male sterile mutant, dysfunctional tapetum1 ( dyt1 ). The dyt1 mutant exhibits abnormal anther morphology beginning at anther stage 4, with tapetal cells that have excess and/or enlarged vacuoles and lack the densely stained cytoplasm typical of normal tapetal cells. The mutant meiocytes are able to complete meiosis I,but they do not have a thick callose wall; they often fail to complete meiotic cytokinesis and eventually collapse. DYT1 encodes a putative bHLH transcription factor and is strongly expressed in the tapetum from late anther stage 5 to early stage 6, and at a lower level in meiocytes. In addition, the level of DYT1 mRNA is reduced in the sporocyteless/nozzle ( spl/nzz ) and excess microsporocytes1/extra sporogenous cell ( ems1/exs ) mutants; together with the mutant phenotypes, this suggests that DYT1 acts downstream of SPL/NZZ and EMS1/EXS . RT-PCR results showed that the expression levels of many tapetum-preferential genes are reduced significantly in the dyt1 mutant, indicating that DYT1 is important for the expression of tapetum genes. Our results support the hypothesis that DYT1 is a crucial component of a genetic network that controls anther development and function.

In higher plants, double fertilisation initiates seed development. One sperm cell fuses with the egg cell and gives rise to the embryo, the second sperm cell fuses with the central cell and gives rise to the endosperm. The endosperm develops as a syncytium with the gradual organisation of domains along an anteroposterior axis defined by the position of the embryo at the anterior pole and by the attachment to the placenta at the posterior pole. We report that ontogenesis of the posterior pole in Arabidopsis thaliana involves oriented migration of nuclei in the syncytium. We show that this migration is impaired in mutants of the three founding members of the FERTILIZATION INDEPENDENT SEED ( FIS ) class, MEDEA ( MEA ), FIS2 and FERTILIZATION INDEPENDENT ENDOSPERM ( FIE ). A screen based on a green fluorescent protein (GFP) reporter line allowed us to identify two new loci in the FIS pathway, medicis and borgia . We have cloned the MEDICIS gene and show that it encodes the Arabidopsis homologue of the yeast WD40 domain protein MULTICOPY SUPRESSOR OF IRA (MSI1). The mutations at the new fis loci cause the same cellular defects in endosperm development as other fis mutations, including parthenogenetic development, absence of cellularisation, ectopic development of posterior structures and overexpression of the GFP marker.

Reproduction in angiosperms depends on communication processes of the male gametophyte (pollen) with the female floral organs (pistil, transmitting tissue) and the female gametophyte (embryo sac). Pollen-pistil interactions control pollen hydration, germination and growth through the stylar tissue. The female gametophyte is involved in guiding the growing pollen tube towards the micropyle and embryo sac. One of the two synergids flanking the egg cell starts to degenerate and becomes receptive for pollen tube entry. Pollen tube growth arrests and the tip of the pollen tube ruptures to release the sperm cells. Failures in the mutual interaction between the synergid and the pollen tube necessarily impair fertility. But the control of pollen tube reception is not understood. We isolated a semisterile, female gametophytic mutant from Arabidopsis thaliana , named feronia after the Etruscan goddess of fertility, which impairs this process. In the feronia mutant, embryo sac development and pollen tube guidance were unaffected in all ovules, although one half of the ovules bore mutant female gametophytes. However, when the pollen tube entered the receptive synergid of a feronia mutant female gametophyte, it continued to grow, failed to rupture and release the sperm cells, and invaded the embryo sac. Thus, the feronia mutation disrupts the interaction between the male and female gametophyte required to elicit these processes. Frequently, mutant embryo sacs received supernumerary pollen tubes. We analysed feronia with synergid-specific GUS marker lines, which demonstrated that the specification and differentiation of the synergids was normal. However, GUS expression in mutant gametophytes persisted after pollen tube entry, in contrast to wild-type embryo sacs where it rapidly decreased. Apparently, the failure in pollen tube reception results in the continued expression of synergid-specific genes, probably leading to an extended expression of a potential pollen tube attractant.

The spindle plays a central role in chromosome segregation during mitosis and meiosis. In particular, various kinesins are thought to play crucial roles in spindle structure and function in both mitosis and meiosis of fungi and animals. A group of putative kinesins has been previously identified in Arabidopsis , called ATK1-ATK4 (previously known as KATA-KATD), but their in vivo functions have not been tested with genetic studies. We report here the isolation and characterization of a mutant, atk1-1, which has a defective ATK1 gene. The atk1-1 mutant was identified in a collection of Ds transposon insertion lines by its reduced fertility. Reciprocal crosses between the atk1-1 mutant and wild type showed that only male fertility was reduced, not female fertility. Molecular analyses, including revertant studies, indicated that the Ds insertion in the ATK1 gene was responsible for the fertility defect. Light microscopy revealed that, in the atk1-1 mutant, male meiosis was defective, producing an abnormal number of microspores of variable sizes. Further cytological studies indicated that meiotic chromosome segregation and spindle organization were both abnormal in the mutant. Specifically, the atk1-1 mutant male meiotic cells had spindles that were broad, unfocused and multi-axial at the poles at metaphase I, unlike the typical fusiform bipolar spindle found in the wild-type metaphase I cells. Therefore, the ATK1 gene plays a crucial role in spindle morphogenesis in male Arabidopsis meiosis.