LACHESIS Restricts Gametic Cell Fate in the Female Gametophyte of Arabidopsis

In flowering plants, the egg and sperm cells form within haploid gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg cell and the central cell, which are flanked by five accessory cells. Both gametic and accessory cells are vital for fertilization; however, the mechanisms that underlie the formation of accessory versus gametic cell fate are unknown. In a screen for regulators of egg cell fate, we isolated the lachesis (lis) mutant which forms supernumerary egg cells. In lis mutants, accessory cells differentiate gametic cell fate, indicating that LIS is involved in a mechanism that prevents accessory cells from adopting gametic cell fate. The temporal and spatial pattern of LIS expression suggests that this mechanism is generated in gametic cells. LIS is homologous to the yeast splicing factor PRP4, indicating that components of the splice apparatus participate in cell fate decisions.     

LACHESIS restricts gametic cell fate in the female gametophyte of Arabidopsis

In flowering plants, the egg and sperm cells form within haploid gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg cell and the central cell, which are flanked by five accessory cells. Both gametic and accessory cells are vital for fertilization; however, the mechanisms that underlie the formation of accessory versus gametic cell fate are unknown. In a screen for regulators of egg cell fate, we isolated the lachesis (lis) mutant which forms supernumerary egg cells. In lis mutants, accessory cells differentiate gametic cell fate, indicating that LIS is involved in a mechanism that prevents accessory cells from adopting gametic cell fate. The temporal and spatial pattern of LIS expression suggests that this mechanism is generated in gametic cells. LIS is homologous to the yeast splicing factor PRP4, indicating that components of the splice apparatus participate in cell fate decisions.     

Female gametophytic cell specification and seed development require the function of the putative Arabidopsis INCENP ortholog WYRD

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.     

Mutants with aberrant numbers of gametic cells shed new light on old questions

In contrast to animals, plant gametes form in distinct haploid generations, termed gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg and central cell, which are flanked by accessory cells. The gametic cells differ with respect to morphology, molecular attributes and, importantly, their fate: whereas the egg cell, upon fertilisation, gives rise to the embryo, the central cell forms the endosperm. To ensure correct endosperm formation, not only the egg cell but also the central cell has to fuse with a sperm cell. The respective sperm cell pair is delivered by a single pollen tube. In some plant species, the two male gametes appear to express a different bias towards the female gametes. Such a preference consequently determines their respective contribution to either embryo or endosperm development. In Arabidopsis and many other species the sperm cells are indistinguishable and it has been discussed whether they possess an inherent preference for either of the female gametes. The recent isolation of mutants that form an aberrant number of either male or female gametes stimulates discussion, albeit with different results. Furthermore, some data indicate that the central cell is competent to initiate endosperm formation without a paternal contribution. These data support the theory that the endosperm is of gametophytic rather than sporophytic origin.     

A dynamic reciprocal RBR-PRC2 regulatory circuit controls Arabidopsis gametophyte development

Unlike animals that produce gametes upon differentiation of meiotic products, plants develop haploid male and female gametophytes that differentiate gametes such as sperm, egg and central cells, and accessory cells [1, 2]. Both gametophytes participate in double fertilization and give rise to the next sporophytic generation. Little is known about the function of cell-cycle genes in differentiation and development of gametophytes and in reproduction [1, 2]. RETINOBLASTOMA RELATED (RBR) is a plant homolog of the tumor suppressor Retinoblastoma (pRb), which is primarily known as negative regulator of the cell cycle [3]. We show that RBR is required for cell differentiation of male and female gametophytes in Arabidopsis and that loss of RBR perturbs expression levels of the evolutionarily ancient Polycomb Repressive Complex 2 (PRC2) subunits and their modifiers encoding PRC2 subunits or DNA METHYLTRANSFERASE 1 (MET1) [4-6], exemplifying convergent evolution involving the RBR-PRC2-MET1 regulatory pathways. In addition, RBR binds MET1, and maintenance of heterochromatin in central cells, a mechanism that is likely mediated by MET1[7, 8], is impaired in the absence of RBR. Surprisingly, PRC2-specific H3K27-trimethylation activity represses paternal RBR allele, suggesting a functional role for a dynamic and reciprocal RBR-PRC2 regulatory circuit in cellular differentiation and reproductive development.     

GAMETOPHYTIC FACTOR 1, Involved in Pre-mRNA Splicing, Is Essential for Megagametogenesis and Embryogenesis in Arabidopsis

RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 (GFA1); a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homoiogs of gene GFA1 in yeast and human encode putative U5 snRNPspecific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogeneeis and embryogenesis in plant.     

GNOM-LIKE 2, Encoding an Adenosine Diphosphate-Ribosylation Factor-Guanine Nucleotide Exchange Factor Protein Homologous to GNOM and GNL1, is Essential for Pollen Germination in Arabidopsis

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 ( gnl2-1 ) and gnl2-2 in Arabidopsis thaliana , in which the pollen grains failed to germinate in vitro and in vivo . GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.     

Mitochondrial GFA2 is required for synergid cell death in Arabidopsis

Little is known about the molecular processes that govern female gametophyte (FG) development and function, and few FG-expressed genes have been identified. We report the identification and phenotypic analysis of 31 new FG mutants in Arabidopsis. These mutants have defects throughout development, indicating that FG-expressed genes govern essentially every step of FG development. To identify genes involved in cell death during FG development, we analyzed this mutant collection for lines with cell death defects. From this analysis, we identified one mutant, gfa2, with a defect in synergid cell death. Additionally, the gfa2 mutant has a defect in fusion of the polar nuclei. We isolated the GFA2 gene and show that it encodes a J-domain-containing protein. Of the J-domain-containing proteins in Saccharomyces cerevisiae (budding yeast), GFA2 is most similar to Mdj1p, which functions as a chaperone in the mitochondrial matrix. GFA2 is targeted to mitochondria in Arabidopsis and partially complements a yeast mdj1 mutant, suggesting that GFA2 is the Arabidopsis ortholog of yeast Mdj1p. These data suggest a role for mitochondria in cell death in plants.     

Female gametophytic mutants of Arabidopsis thaliana identified in a gene trap insertional mutagenesis screen

In plants, the male and female gametophytes represent the haploid generation that alternates with the diploid sporophytic generation. Male and female gametophytes develop from haploid micro- and megaspores, respectively. In flowering plants (angiosperms), the spores themselves arise from the sporophyte through meiotic divisions of sporogenous cells in the reproductive organs of the flower. Male and female gametophytes contain two pairs of gametes that participate in double fertilization, a distinctive feature of angiosperms. In this paper, we describe the employment of a transposon-based gene trap system to identify mutations affecting the gametophytic phase of the plant life cycle. Mutants affecting female gametogenesis were identified in a two-step screen for (i) reduced fertility (seed abortion or undeveloped ovules) and (ii) segregation ratio distortion. Non-functional female gametophytes do not initiate seed development, leading to semi-sterility such that causal or linked alleles are transmitted at reduced frequency to the progeny (non-Mendelian segregation). From a population of 2,511 transposants, we identified 54 lines with reduced seed set (2%). Examination of their distorted segregation ratios and seed phenotypes led to the isolation of 12 gametophytic mutants, six of which are described herein. Chromosomal sequences flanking the transposon insertions were identified and physically mapped onto the genome sequence of Arabidopsis thaliana. Surprisingly, the insertion sites were often associated with chromosomal rearrangements, making it difficult to assign the mutant phenotypes to a specific gene. The mutants were classified according to the process affected at the time of arrest, i.e. showing mitotic, karyogamic, maternal or degenerative phenotypes.     

An Arabidopsis aspartic protease functions as an anti-cell-death component in reproduction and embryogenesis

The components and pathways that regulate and execute developmental cell death programmes in plants remain largely unknown. We have found that the PROMOTION OF CELL SURVIVAL 1 (PCS1) gene in Arabidopsis, which encodes an aspartic protease, has an important role in determining the fate of cells in embryonic development and in reproduction processes. The loss-of-function mutation of PCS1 causes degeneration of both male and female gametophytes and excessive cell death of developing embryos. Conversely, ectopic expression of PCS1 causes the septum and stomium cells that normally die in the anther wall to survive instead, leading to a failure in anther dehiscence and male sterility. PCS1 provides a new avenue for understanding the mechanisms of the programmed cell death processes that are associated with developmental pathways in plants and makes available a useful tool for engineering the male sterility trait for hybrid seed production.     

A null mutation of ROS1a for DNA demethylation in rice is not transmittable to progeny

Genes that promote DNA methylation and demethylation in plants have been characterized mainly in Arabidopsis. Arabidopsis DNA demethylation is mediated by bi-functional DNA enzymes with glycosylase activity that removes 5-methylcytosine and lyase activity that nicks double-stranded DNA at an abasic site. Homologous recombination-promoted knock-in targeting of the ROS1a gene, the longest of six putative DNA demethylase genes in the rice genome, by fusing its endogenous promoter to the GUS reporter gene, led to reproducibly disrupted ROS1a in primary (T(0)) transgenic plants in the heterozygous condition. These T(0) plants exhibited no overt morphological phenotypes during the vegetative phase, and GUS staining showed ROS1a expression in pollen, unfertilized ovules and meristematic cells. Interestingly, neither the maternal nor paternal knock-in null allele, ros1a-GUS1, was virtually detected in the progeny; such an intransmittable null mutation is difficult to isolate by conventional mutagenesis techniques that are usually used to identify and isolate mutants in the progeny population. Even in the presence of the wild-type paternal ROS1a allele, the maternal ros1a-GUS1 allele caused failure of early-stage endosperm development, resulting in incomplete embryo development, with embryogenesis producing irregular but viable embryos that failed to complete seed dormancy, implying non-equivalent maternal and paternal contribution of ROS1a in endosperm development. The paternal ros1a-GUS1 allele was not transmitted to progeny, presumably because of a male gametophytic defect(s) prior to fertilization. Thus, ROS1a is indispensable in both male and female gametophytes, and DNA demethylation must plays important roles in both gametophytes.     

Segregation distortion in Arabidopsis <Emphasis Type="Italic">gametophytic factor 1 </Emphasis>(<Emphasis Type="Italic">gfa1</Emphasis>) mutants is caused by a deficiency of an essential RNA splicing factor

The female and male gametophytes are critical components of the angiosperm life cycle and are essential for the reproductive process. The gametophytes share many essential cellular processes with each other and with the sporophyte generation. As a consequence, these processes can only be analyzed genetically in the gametophyte generation. Here, we report the characterization of the gametophytic factor 1 (gfa1) mutant. The gfa1 mutation exhibits reduced transmission through both the female and male gametophytes. Reduced transmission through the female gametophyte is due to an effect on female gametophyte development. By contrast, development of the pollen grain is not affected in gfa1; rather, reduced transmission is likely due to an effect on pollen tube growth. We have identified multiple T-DNA-insertion alleles of gfa1 in a gene encoding a protein with high similarity to Snu114/U5-116 kD proteins from yeast and animals required for normal pre-mRNA splicing. Consistent with its predicted function, the GFA1 gene (At1g06220) is expressed throughout the plant. Together, these data suggest that GFA1 functions in mRNA splicing during the plant life cycle. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Development of male gametes in flowering plants

The male gametes of angiosperms consist of two sperm cells within a pollen grain or a pollen tube. They are derived from a single generative cell, which is formed as the smaller cell by unequal cell division in the microspore after meiosis. Limited information is available about these male gametic cells, beyond observations by electron microscopy, because each is surrounded by the cytoplasm of a larger vegetative cell. Recently, large quantities of generative cells and sperm cells have been isolated from pollen grains or pollen tubes of various plant species, and their physiological, biochemical and molecular characterization is now possible. Although almost all the available results are still preliminary, it is evident that the male gametic cells are peculiar in terms both of cell structure and composition. For example, they are rich in axial microtubules which maintain the spindle-like shape of each cell. However, they lack plastids which are DNA-containing cytoplasmic organelles. Biochemical characterization of their proteins indicates the presence of male gamete-specific polypeptides. These findings suggest, not unexpectedly, the possibility of male gamete-specific gene expression and of a strict genetic mechanism that controls the formation of male gametes.     

Targeted degradation of the cyclin-dependent kinase inhibitor ICK4/KRP6 by RING-type E3 ligases is essential for mitotic cell cycle progression during Arabidopsis gametogenesis

Following meiosis, plant gametophytes develop through two or three rounds of mitosis. Although the ontogeny of gametophyte development has been defined in Arabidopsis thaliana, the molecular mechanisms regulating mitotic cell cycle progression are not well understood. Here, we report that RING-H2 group F 1a (RHF1a) and RHF2a, two RING-finger E3 ligases, play an important role in Arabidopsis gametogenesis. The rhf1a rhf2a double mutants are defective in the formation of male and female gametophytes due to interphase arrest of the mitotic cell cycle at the microspore stage of pollen development and at female gametophyte stage 1 of embryo sac development. We demonstrate that RHF1a directly interacts with and targets a cyclin-dependent kinase inhibitor ICK4/KRP6 (for Interactors of Cdc2 Kinase 4/Kip-related protein 6) for proteasome-mediated degradation. Inactivation of the two redundant RHF genes leads to the accumulation of ICK4/KRP6, and reduction of ICK4/KRP6 expression largely rescues the gametophytic defects in rhf1a rhf2a double mutants, indicating that ICK4/KRP6 is a substrate of the RHF E3 ligases. Interestingly, in situ hybridization showed that ICK4/KRP6 was predominantly expressed in sporophytes during meiosis. Our findings indicate that RHF1a/2a-mediated degradation of the meiosis-accumulated ICK4/KRP6 is essential to ensure the progression of subsequent mitoses to form gametophytes in Arabidopsis.     

The resurgence of haploids in higher plants

The life cycle of plants proceeds via alternating generations of sporophytes and gametophytes. The dominant and most obvious life form of higher plants is the free-living sporophyte. The sporophyte is the product of fertilization of male and female gametes and contains a set of chromosomes from each parent; its genomic constitution is 2n. Chromosome reduction at meiosis means cells of the gametophytes carry half the sporophytic complement of chromosomes (n). Plant haploid research began with the discovery that sporophytes can be produced in higher plants carrying the gametic chromosome number (n instead of 2n) and that their chromosome number can subsequently be doubled up by colchicine treatment. Recent technological innovations, greater understanding of underlying control mechanisms and an expansion of end-user applications has brought about a resurgence of interest in haploids in higher plants.