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.     

Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey

One major player known to be essential for successful gamete interactions during double fertilization in Arabidopsis thaliana is the recently identified family of egg cell-secreted EC1 proteins. Both gamete fusion events are affected in EC1-deficient female gametophytes. Here, we show that the number of ovules with unfused sperm cells is considerably higher than the number of undeveloped seeds in the same ec1-RNAi knockdown lines. We found that some sperm cells are able to fuse with the female gametes even 2 to 3 days after pollination, as reflected by delayed embryo and endosperm development, and by polytubey. We propose that the egg cell secretes EC1 proteins upon sperm arrival to promote rapid sperm activation, thereby accelerating gamete fusion and preventing polytubey.     

Isolation of Viable Male and Female Gametes in Cunninghamia lanceolata

Viable male and female gametes were isolated from pollinated ovules of Cunninghamia lanceolata (Lamb.) Hook. Prior to their penetration into the female gametophyte, the pollen tubes were drawn out from the nucetlus. The isolated pollen tubes were branched and one of them became swollen. An enlarged spermatogenous cell and subsquently a pair of sperm cells were formed as the pollen tube reached the regions over and against the archegonia. The sperm cells were released from the pollen tubes manually with the use of a stereomicroscope. The positive FDA reaction gave evidence of the sperm cells viability, and the Fluorescent Brightener 28 positive demonstrated the presence of cell wall. The egg cells were enzymatically isolated from the female gametophytes. The isolated egg cells were spherical, contained 1 to 2 large and many small vacuoles. FDA test showed the egg cells were viable, and the viability sustained for 8 days in 2 to 4 ℃ without any protectants. Fusion between single pair of male and female gametic protoplasts was attempted with PEG method, but only adhesion of the two was obtained.     

An Efficient Method for Quantitative,Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.     

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.     

Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule

In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.     

雌、雄配子体间及雌配子体成员细胞间的通讯

被子植物的双受精是一个复杂而精密的调控过程.成功的受精依赖于配子体的正确发育以及雌配子体与雄配子体间的相互识别.研究表明,雌配子体自身成员细胞间存在广泛的胞间通讯.这种通讯不仅影响不同细胞的发育进程,也决定细胞的发育命运,从而保证雌配子体的正常发育.此外,雌配子体与雄配子体间存在胞间通讯,这种胞间通讯是雌配子体与雄配子体间相互识别的分子基础,精确调控了雄配子体准确进入珠孔、在雌配子体内适时停止伸长、尖端破裂并在特定位置释放精细胞等过程.本文概述了这些方面的最新进展,梳理胞间通讯的途径与信号,并展望了未来雌、雄配子体间及雌配子体成员细胞间通讯的研究方向与可能的应用前景.     

A role for LORELEI,a putative glycosylphosphatidylinositol‐anchored protein,in Arabidopsis thaliana double fertilization and early seed development

In plants, double fertilization requires successful sperm cell delivery into the female gametophyte followed by migration, recognition and fusion of the two sperm cells with two female gametes. We isolated a null allele (lre‐5) of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)‐anchored protein implicated in reception of the pollen tube by the female gametophyte. Although most lre‐5 female gametophytes do not allow pollen tube reception, in those that do, early seed development is delayed. A fraction of lre‐5/lre‐5 seeds underwent abortion due to defect(s) in the female gametophyte. The aborted seeds contained endosperm but no zygote/embryo, reminiscent of autonomous endosperm development in the pollen tube reception mutants scylla and sirene. However, unpollinated lre‐5/lre‐5 ovules did not initiate autonomous endosperm development and endosperm development in aborted seeds began after central cell fertilization. Thus, the egg cell probably remained unfertilized in aborted lre‐5/lre‐5 seeds. The lre‐5/lre‐5 ovules that remain undeveloped due to defective pollen tube reception did not induce synergid degeneration and repulsion of supernumerary pollen tubes. In ovules, LORELEI is expressed during pollen tube reception, double fertilization and early seed development. Null mutants of LORELEI‐like‐GPI‐anchored protein 1 (LLG1), the closest relative of LORELEI among three Arabidopsis LLG genes, are fully fertile and did not enhance reproductive defects in lre‐5/lre‐5 pistils, suggesting that LLG1 function is not redundant with that of LORELEI in the female gametophyte. Our results show that, besides pollen tube reception, LORELEI also functions during double fertilization and early seed development.     

The Polycomb group protein MEDEA and the DNA methyltransferase MET1 interact to repress autonomous endosperm development in Arabidopsis

In flowering plants, double fertilization of the female gametes, the egg and the central cell, initiates seed development to give rise to a diploid embryo and the triploid endosperm. In the absence of fertilization, the FERTILIZATION‐INDEPENDENT SEED Polycomb Repressive Complex 2 (FIS‐PRC2) represses this developmental process by histone methylation of certain target genes. The FERTILIZATION‐INDEPENDENT SEED (FIS) class genes MEDEA (MEA) and FERTILIZATION‐INDEPENDENT ENDOSPERM (FIE) encode two of the core components of this complex. In addition, DNA methylation establishes and maintains the repression of gene activity, for instance via DNA METHYLTRANSFERASE1 (MET1), which maintains methylation of symmetric CpG residues. Here, we demonstrate that Arabidopsis MET1 interacts with MEA in vitro and in a yeast two‐hybrid assay, similar to the previously identified interaction of the mammalian homologues DNMT1 and EZH2. MET1 and MEA share overlapping expression patterns in reproductive tissues before and after fertilization, a prerequisite for an interaction in vivo. Importantly, a much higher percentage of central cells initiate endosperm development in the absence of fertilization in mea‐1/MEA; met1‐3/MET1 as compared to mea‐1/MEA mutant plants. In addition, DNA methylation at the PHERES1 and MEA loci, imprinted target genes of the FIS‐PRC2, was affected in the mea‐1 mutant compared with wild‐type embryos. In conclusion, our data suggest a mechanistic link between two major epigenetic pathways involved in histone and DNA methylation in plants by physical interaction of MET1 with the FIS‐PRC2 core component MEA. This concerted action is relevant for the repression of seed development in the absence of fertilization.     

The Arabidopsis mutant feronia disrupts the female gametophytic control of pollen tube reception

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.     

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.     

Isolation of gametes and central cells from Oryza sativa L.

In vitro fertilization system of higher plants has been well established using maize gametes and central cells, which can produce embryos and endosperms. In the present study, procedures for isolating gametes and central cells from rice (Oryza sativa L. cv. Nipponbare), a model plant, are reported with the goal of establishing rice in vitro fertilization system. Egg cells and central cells were isolated by manual manipulation of enzyme-treated unpollinated ovules, and an alternative direct isolation method for egg cells that does not use enzymatic treatment was also established. Fluorescent visualization of the granular structures in the cytoplasm of isolated egg cells and the nucleoli in two polar nuclei of isolated central cells suggest that these cells are reliable gametes and central cells. For sperm cell isolation, the contents of rice pollen grains were released by osmotic pressure-induced bursting of the grains. In addition, electrofusion with isolated gametes was successfully conducted.     

The Cell Cycle Timing of Centromeric Chromatin Assembly in Drosophila Meiosis Is Distinct from Mitosis Yet Requires CAL1 and CENP-C

CENP-A (CID in flies) is the histone H3 variant essential for centromere specification, kinetochore formation, and chromosome segregation during cell division. Recent studies have elucidated major cell cycle mechanisms and factors critical for CENP-A incorporation in mitosis, predominantly in cultured cells. However, we do not understand the roles, regulation, and cell cycle timing of CENP-A assembly in somatic tissues in multicellular organisms and in meiosis, the specialized cell division cycle that gives rise to haploid gametes. Here we investigate the timing and requirements for CID assembly in mitotic tissues and male and female meiosis in Drosophila melanogaster, using fixed and live imaging combined with genetic approaches. We find that CID assembly initiates at late telophase and continues during G1 phase in somatic tissues in the organism, later than the metaphase assembly observed in cultured cells. Furthermore, CID assembly occurs at two distinct cell cycle phases during male meiosis: prophase of meiosis I and after exit from meiosis II, in spermatids. CID assembly in prophase I is also conserved in female meiosis. Interestingly, we observe a novel decrease in CID levels after the end of meiosis I and before meiosis II, which correlates temporally with changes in kinetochore organization and orientation. We also demonstrate that CID is retained on mature sperm despite the gross chromatin remodeling that occurs during protamine exchange. Finally, we show that the centromere proteins CAL1 and CENP-C are both required for CID assembly in meiosis and normal progression through spermatogenesis. We conclude that the cell cycle timing of CID assembly in meiosis is different from mitosis and that the efficient propagation of CID through meiotic divisions and on sperm is likely to be important for centromere specification in the developing zygote.     

Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana

Arabidopsis has three cytokinin receptors genes: CRE1, AHK2 and AHK3. Availability of plants that are homozygous mutant for these three genes indicates that cytokinin receptors in the haploid cells are dispensable for the development of male and female gametophytes. The triple mutants form a few flowers but never set seed, indicating that reproductive growth is impaired. We investigated which reproductive processes are affected in the triple mutants. Anthers of mutant plants contained fewer pollen grains and did not dehisce. Pollen in the anthers completed the formation of the one vegetative nucleus and the two sperm nuclei, as seen in wild type. The majority of the ovules were abnormal: 78% lacked the embryo sac, 10% carried a female gametophyte that terminated its development before completing three rounds of nuclear division, and about 12% completed three rounds of nuclear division but the gametophytes were smaller than those of the wild type. Reciprocal crosses between the wild type and the triple mutants indicated that pollen from mutant plants did not germinate on wild-type stigmas, and wild-type pollen did not germinate on mutant stigmas. These results suggest that cytokinin receptors in the sporophyte are indispensable for anther dehiscence, pollen maturation, induction of pollen germination by the stigma and female gametophyte formation and maturation.Key words: cytokinin, cytokinin receptor, female gametophyte, male gametophyte, stigma     

烟草雌、雄配子DNA 含量变化

采用显微分光光度法测定了烟草( Nicotiana tabacum) 精细胞和卵细胞的DNA 含量。烟草是二胞花粉, 花粉萌发后生殖细胞在花粉管中分裂形成精细胞。授粉后45 h 花粉管到达子房, 在花粉管内的精细胞DNA 含量为1C。当花粉管在退化助细胞中破裂, 释放出的两个精细胞开始合成DNA。在与卵细胞融合前,两个精细胞DNA 含量接近2C。随着精细胞的到达及合成DNA, 卵细胞也开始合成DNA, 融合前的卵细胞DNA 含量也接近2C。精、卵细胞融合后, 合子DNA 含量为4C。烟草雌、雄配子是在细胞周期的G2 期发生融合, 属于G2 型。