New insights into the functional roles of reactive oxygen species during embryo sac development and fertilization in Arabidopsis thaliana

Previously considered as toxic by-products of aerobic metabolism, reactive oxygen species (ROS) are emerging as essential signaling molecules in eukaryotes. Recent evidence showed that maintenance of ROS homeostasis during female gametophyte development is crucial for embryo sac patterning and fertilization. Although ROS are exclusively detected in the central cell of mature embryo sacs, the study of mutants deficient in ROS homeostasis suggests that controlled oxidative bursts might take place earlier during gametophyte development. Also, a ROS burst that depends on pollination takes place inside the embryo sac. This oxidative response might be required for pollen tube growth arrest and for sperm cell release. In this mini-review, we will focus on new insights into the role of ROS during female gametophyte development and fertilization. Special focus will be made on the mitochondrial Mn-Superoxide dismutase (MSD1), which has been recently reported to be essential for maintaining ROS homeostasis during embryo sac formation.     

Embryo Sac Development in the Maize indeterminate gametophyte1 Mutant: Abnormal Nuclear Behavior and Defective Microtubule Organization

The indeterminate gametophyte1 mutation in maize has been known to disrupt development of the female gametophyte. Mutant embryo sacs have abnormal numbers and behavior of micropylar and central cell nuclei, which result in polyembryony and elevated ploidy levels in the endosperm of developing kernels. In this study, we confirm abnormal nuclear behavior and present novel findings. In contrast to the normal form, there is no obvious polarity in two-nucleate embryo sacs or in the micropylar cells of eight-nucleate embryo sacs. We show that the second and third mitoses are not fully synchronized and that additional mitoses can occur in all of the nuclei of the mutant embryo sac or in just the micropylar or central regions. After cellularization, individual micropylar cells can undergo mitosis. Abnormal microtubular behavior results in irregular positioning of the nuclei, asynchronous microtubular patterns in different pairs of nuclei, and abnormal phragmoplasts after the third mitotic division. These results indicate that in addition to acting primarily in controlling nuclear divisions, the indeterminate gametophyte1 gene acts secondarily in regulating microtubule behavior. This cytoskeletal activity most likely controls the polarization and nuclear migration underlying the formation and fate of the cells of the normal embryo sac.     

The NOMEGA gene required for female gametophyte development encodes the putative APC6/CDC16 component of the Anaphase Promoting Complex in Arabidopsis

Development of the female gametophyte involves several rounds of nuclear divisions during which nuclei are rearranged and finally cellularized to form a mature seven-celled embryo sac. During these nuclear divisions, key proteins involved in the cell cycle need to be degraded quickly in order to facilitate both the metaphase-anaphase transition stage and late anaphase. Here, we report the characterization of an Arabidopsis mutant nomega, which results in arrest of the embryo sac development at the two-nucleate stage. The NOMEGA gene product shows high homology to the APC6/cell division cycle (CDC)16 subunit of the Anaphase Promoting Complex/Cyclosome (APC/C). The phenotype of the nomega mutant is quite different from that of the hobbit mutant, which had suggested a role for the plant APC/C in auxin signalling. We show that nomega mutant embryo sacs are unable to degrade Cyclin B, an important APC/C substrate, providing further evidence of a role for the NOMEGA gene product and the plant APC/C in cell cycle progression during gametophyte development.     

矮沙冬青雌配子体及胚胎发育研究

矮沙冬青子房单心皮1室,边缘胎座,弯生胚珠,胚珠具双珠被、厚珠心。大孢子孢原细胞发生于珠心表皮下,大孢子母细胞减数分裂形成直线排列的四分体,合点端大孢子具功能,并按蓼型胚囊发育,雌配子体成熟于4月中旬。双受精后,胚乳发育为核型。在矮沙冬青大孢子发生、雌配子体和胚胎发育过程中未发现异常现象,因此认为矮沙冬青濒危不存在雌性生殖结构与发育过程异常的内在因素。     

Proteomics of pollen development and germination

In higher plants, pollen grains represent the vestiges of a highly reduced male gametophyte generation. After germination, the pollen tube delivers the sperm cells by tip-growing to the embryo sac for fertilization. Besides the intrinsic importance for sexual reproduction, pollen development and germination serve as an attractive system to address important questions related to cell division, cell differentiation, polar growth, cell-cell interaction, and cell fate. Recently, pollen functional specification has been well-studied using multidisciplinary approaches. Here, we review recent advances in proteomics of pollen development and germination.     

Unusual microtubular cytoskeleton of apomictic embryo sac of Chondrilla juncea L.

Summary. The mature apomictic embryo sac of Chondrilla juncea is highly vacuolated and demonstrates a polarization similar to that of the amphimictic gametophyte. The microtubule cytoskeleton of this embryo sac is uncharacteristic and relatively weak. The microtubules are positioned along cell walls and resemble cortical microtubules of somatic cells. They do not form the parallel, brushlike structures observed around the filiform apparatus of synergids in the amphimictic embryo sac. In the apomictic embryo sac, the microtubules of both the egg cell and the central cell develop a cortical-like structure, which is entirely different from the radial arrangement observed around the nuclei in the amphimictic embryo sac. Correspondence and reprints: Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31-044 Kraków, Poland.     

The dyad gene is required for progression through female meiosis in Arabidopsis

In higher plants the gametophyte consists of a gamete in association with a small number of haploid cells, specialized for sexual reproduction. The female gametophyte or embryo sac, is contained within the ovule and develops from a single cell, the megaspore which is formed by meiosis of the megaspore mother cell. The dyad mutant of Arabidopsis, described herein, represents a novel class among female sterile mutants in plants. dyad ovules contain two large cells in place of an embryo sac. The two cells represent the products of a single division of the megaspore mother cell followed by an arrest in further development of the megaspore. We addressed the question of whether the division of the megaspore mother cell in the mutant was meiotic or mitotic by examining the expression of two markers that are normally expressed in the megaspore mother cell during meiosis. Our observations indicate that in dyad, the megaspore mother cell enters but fails to complete meiosis, arresting at the end of meiosis 1 in the majority of ovules. This was corroborated by a direct observation of chromosome segregation during division of the megaspore mother cell, showing that the division is a reductional and not an equational one. In a minority of dyad ovules, the megaspore mother cell does not divide. Pollen development and male fertility in the mutant is normal, as is the rest of the ovule that surrounds the female gametophyte. The embryo sac is also shown to have an influence on the nucellus in wild type. The dyad mutation therefore specifically affects a function that is required in the female germ cell precursor for meiosis. The identification and analysis of mutants specifically affecting female meiosis is an initial step in understanding the molecular mechanisms underlying early events in the pathway of female reproductive development.     

Auxin Import and Local Auxin Biosynthesis Are Required for Mitotic Divisions,Cell Expansion and Cell Specification during Female Gametophyte Development in Arabidopsis thaliana

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.     

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.     

Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1

In Arabidopsis thaliana, the female gametophyte is a highly polarized structure consisting of four cell types: one egg cell and two synergids, one central cell, and three antipodal cells. In this report, we describe the characterization of a novel female gametophyte mutant, eostre, which affects establishment of cell fates in the mature embryo sac. The eostre phenotype is caused by misexpression of the homeodomain gene BEL1-like homeodomain 1 (BLH1) in the embryo sac. It is known that BELL-KNAT proteins function as heterodimers whose activities are regulated by the Arabidopsis ovate family proteins (OFPs). We show that the phenotypic effect of BLH1 overexpression is dependent upon the class II knox gene KNAT3, suggesting that KNAT3 must be expressed and functional during megagametogenesis. Moreover, disruption of At OFP5, a known interactor of KNAT3 and BLH1, partially phenocopies the eostre mutation. Our study indicates that suppression of ectopic activity of BELL-KNOX TALE complexes, which might be mediated by At OFP5, is essential for normal development and cell specification in the Arabidopsis embryo sac. As eostre-1 embryo sacs also show nuclear migration abnormalities, this study suggests that a positional mechanism might be directing establishment of cell fates in early megagametophyte development.     

Actin cytoskeleton in the extra-ovular embryo sac of Utricularia nelumbifolia (Lentibulariaceae)

The actin cytoskeleton in the mature female gametophyte of angiosperms has been examined in only a few dicot and monocot species. The main purposes of this study were to identify how the actin cytoskeleton is arranged in the mature extra-ovular embryo sac in Utricularia nelumbifolia (Lentibulariaceae). We found that the extra-ovular part of the central cell has a well-developed actin cytoskeleton: actin microfilaments formed of long strands which run longitudinally or transversally to the long axis of the embryo sac. The exerted part of the central cell, which is exposed to the environment of the ovary chamber, is highly vacuolated and in the thin peripheral cytoplasm possesses a complicated network of actin microfilaments. The epidermal cells of the placenta that are in contact with the extra-ovular part of the embryo sac are crushed. The ultrastructure data of these cells are presented. We detected the accumulation of the actin cytoskeleton between the micropylar parts of the synergids and the extra-ovular part of central cell. This actin accumulation is unusual because in typical angiosperms the micropylar parts of the synergids form the apex of the female gametophyte.     

Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indica/japonica Hybrids in Rice

Embryo sac abortion is one of the major reasons for sterility in indica/japonica hybrids in rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indica/japonica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagametogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucellus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.     

Abnormalities Occurring during Female Gametophyte Development Result in the Diversity of Abnormal Embryo Sacs and Leads to Abnormal Fertilization in indicaljaponica Hybrids in Rice

Embryo sac abortion is one of the major masons for sterility in indicaljaponica hybrids In rice. To clarify the causal mechanism of embryo sac abortion, we studied the female gametophyte development in two indicaljaponica hybrids via an eosin B staining procedure for embryo sac scanning using confocal laser scanning microscope. Different types of abnormalities occurred during megasporogenesis and megagamatogenesis were demonstrated. The earliest abnormality was observed in the megasporocyte. A lot of the chalazal-most megaspores were degenerated before the mono-nucleate embryo sac stage. Disordered positioning of nucleus and abnormal nucallus tissue were characteristics of the abnormal female gametes from the mono-nucleate to four-nucleate embryo sac stages. The abnormalities that occurred from the early stage of the eight-nucleate embryo sac development to the mature embryo sac stage were characterized by smaller sizes and wrinkled antipodals. Asynchronous nuclear migration, abnormal positioning of nucleus, and degeneration of egg apparatus were also found at the eight-nucleate embryo sac stage. The abnormalities that occurred during female gametophyte development resulted in five major types of abnormal embryo sacs. These abnormal embryo sacs led to abnormal fertilization. Hand pollination using normal pollens on the spikelets during anthesis showed that normal pollens could not exclude the effect of abnormal embryo sac on seed setting.     

大花君子兰大小孢子发生与雌雄配子体发育的研究

该研究运用常规石蜡切片技术,对大花君子兰(Clivia miniata Regel)大、小孢子发生及雌、雄配子体发育进程进行解剖学观察分析,以探讨君子兰生殖生物学解剖特征,为君子兰种子发育和育种提供理论依据。结果表明：(1)大花君子兰花药4室,具分泌型绒毡层。(2)小孢子母细胞减数分裂的胞质分裂为连续型,小孢子四分体为左右对称型,成熟花粉为二细胞型。(3)倒生胚珠,双珠被,厚珠心和雌配子体发育为蓼型。(4)记录了雌雄配子体发育的对应关系,发现雄配子体发育趋于同步,雌配子体发育不同步。(5)开花散粉时,雌配子体尚有处于四核、八核胚囊的时期;成熟胚囊阶段,中央细胞的2个极核位于反足细胞端,反足细胞呈退化状态。具承珠盘结构。     

Development of the Female Gametophyte in Hydrobryum griffithii (Podostemaceae)

The development of the female gametophyte in Hydrobryum griffithiiis of the Apinagia type. The chalazal megaspore nucleus of thetwo-nucleate embryo sac completely degenerates, and only themicropylar megaspore nucleus contributes to the four nucleipresent in the organized embryo sac. The female gametophyteconsists of two synergids, an egg and a haploid central cell.The latter degenerates before the entry of the pollen tube andthere is only syngamy. The nucellar cells below the embryo sacorganize into a nucellar plasmodium.