The putative sensor histidine kinase CKI1 is involved in female gametophyte development in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Embryo sac formation is a fundamental step in sexual reproduction in plants. However, the key players involved in the development of the female gametophyte remain elusive. We present data indicating that a two-component sensor histidine kinase, CKI1, originally implicated in cytokinin perception, is required for completion of megagametogenesis in Arabidopsis. We isolated a loss-of-function mutation in CKI1 resulting from an insertion of the En-1 transposon into the CKI1 coding sequence. Genetic analysis revealed that the mutant allele, cki1-i, could not be transmitted through the female germ line. Confocal laser scanning microscopy identified a block in megagametogenesis, characterized by the abortion of the central vacuole in mutant embryo sacs, and degradation of the developing female gametophyte after completion of all mitotic divisions. The recovery of two independent stable alleles and one revertant wild-type allele resulting from En-1 excision confirmed unambiguously the causal link between the cki1-i mutation and the abnormal phenotype. In situ localization of CKI1 mRNA and histochemical analysis of stable transformants harboring the uidA gene under the control of CKI1 promoter revealed that expression of CKI1 starts at the very beginning of female gametophyte development, and continues until fertilization. This suggests that the developing embryo sac may remain sensitive to signals recognized by CKI1 throughout megagametogenesis. Furthermore, expression of the paternally transmitted CKI1 was detected early after fertilization. The results indicate a role for a two-component signaling system during female gametophyte development, and provide the first evidence that gametophytic expression of a sensor-like molecule is essential for specific processes during megagametogenesis.     

Development and function of central cell in angiosperm female gametophyte

The central cell characterizes the angiosperm female gametophyte (embryo sac or megagametophyte) in that it directly participates in “double fertilization” to initiate endosperm development, a feature distinguishing angiosperm from all other plant taxa. Polygonum‐type central cell is a binucleate cell that, upon fertilization with one of the two sperm cells, forms triploid endosperm to nourish embryo development. Although the formation and the structure of central cell have well been elucidated, the molecular mechanisms for its specification and development remain largely unknown. The central cell plays a critical role in pollen tube guidance during pollination and in endosperm initiation after fertilization. Recently, a group of mutants affecting specific steps of central cell development and function have been identified, providing some clues in understanding these questions. This review summarizes our current knowledge about central cell development and function, and presents overview about hypotheses for its evolution. genesis 48:466–478, 2010. © 2010 Wiley‐Liss, Inc.     

Cross talk between the sporophyte and the megagametophyte during ovule development

In seed plant ovules, the diploid maternal sporophytic generation embeds and sustains the haploid generation (the female gametophyte); thus, two independent generations coexist in a single organ. Many independent studies on Arabidopsis ovule mutants suggest that embryo sac development requires highly synchronized morphogenesis of the maternal sporophyte surrounding the gametophyte, since megagametogenesis is severely perturbed in most of the known sporophytic ovule development mutants. Which are the messenger molecules involved in the haploid–diploid dialogue? And furthermore, is this one way communication or is a feedback cross talk? In this review, we discuss genetic and molecular evidences supporting the presence of a cross talk between the two generations, starting from the first studies regarding ovule development and ending to the recently sporophytic identified genes whose expression is strictly controlled by the haploid gametophytic generation. We will mainly focus on Arabidopsis studies since it is the species more widely studied for this aspect. Furthermore, possible candidate molecules involved in the diploid–haploid generations dialogue will be presented and discussed.     

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.     

ScRALF3, a secreted RALF‐like peptide involved in cell–cell communication between the sporophyte and the female gametophyte in a solanaceous species

Small peptides have been shown to regulate numerous aspects of plant development through cell–cell communication. These signaling events are particularly important during reproduction, regulating gamete development and embryogenesis. Rapid alkalinization factor (RALF)‐like genes, a large gene family that encodes secreted peptides, have specific or ubiquitous expression patterns. Previously, five RALF‐like genes with potential involvement during reproduction were isolated from Solanum chacoense. Here, we show that ScRALF3 is an important peptide regulator of female gametophyte development. Its expression, which is auxin‐inducible, is strictly regulated before and after fertilization. Down‐regulation of ScRALF3 expression by RNA interference leads to the production of smaller fruits that produce fewer seeds, due to improper development of the embryo sacs. Defects include loss of embryo sac nuclei polarization, as well as an increase in asynchronous division, accounting for cellular dysfunctions and premature embryo sac development arrest during megagametogenesis. ScRALF3 is expressed in the sporophytic tissue surrounding the embryo sac, the integument and the nucellus, as revealed by in situ hybridization and GUS staining. As expected for a secreted peptide, fluorescence from an ScRALF3–GFP fusion construct is detected throughout the secretory pathway. Therefore, the ScRALF3 secreted peptide may be directly involved in the regulation of multiple aspects of cell–cell communication between the female gametophyte and its surrounding sporophytic tissue during ovule development.     

Cell-cell communication during double fertilization

Double fertilization in flowering seed plants requires intercellular signaling events between many interacting partners. The four cell types of the seven-celled female gametophyte communicate with each other to establish and maintain their identity. They secrete signaling molecules to guide the male gametophyte and to mediate sperm cell discharge and transport towards the two female gametes (the egg and central cell). After fusion of the gametes, guidance signals have to be removed to prevent polyspermy, embryo and endosperm development is induced generating daughter cells or nuclear regions of a different fate, and cell death is induced in the surrounding ovular cells. Until recently, little was known about the molecular nature of the signaling molecules that are involved in these processes. Now, small secreted proteins and peptides have been identified as prime candidates mediating several of these communication events.     

Confocal microscopy of whole ovules for analysis of reproductive development: the elongate1 mutant affects meiosis II

Analysis of female meiosis (megasporogenesis) and embryo sac development (megagametogenesis) in angiosperms is technically challenging because the cells are enclosed within the nucellus and ovule tissues of the female flower. This is in contrast to male sporogenesis and gametogenesis where development can readily be observed through the easily dissectable developing anthers. Observation of embryo sac development is a particular problem in crassinucellate ovules such as those of maize. To overcome the problems in observing reproductive development, we developed a simple Feulgen staining procedure optimized for use with confocal microscopy to observe reproductive progression in the crassinucellate ovules of maize. The procedure greatly facilitates the observation of nuclei and cell structures of all stages of megasporogenesis and embryo sac development. The high resolution obtained using the technique enabled us to readily visualize chromosomes from individual cells within ovule tissue samples of maize. A propidium iodide staining technique was also used and compared with the Feulgen-based technique. Static cytometry of relative DNA content of individual nuclei was possible using Imaris software on both Feulgen and propidium iodide-stained samples. The techniques also proved successful for the observation of Arabidopsis and Hieracium aurantiacum female gametophyte and seed development, demonstrating the general applicability of the techniques. Using both staining methods, we analysed the maize meiotic mutant elongate1, which produces functional diploid instead of haploid embryo sacs. The precise defect in meiosis from which diploid embryo sacs arise in elongate1 has not previously been reported. We used confocal microscopy followed by static cytometry using Imaris software to show that the defect by which diploid embryo sacs arise in the maize mutant elongate1 is the absence of meiosis II with one of the dyad cells directly initiating megagametogenesis.     

Maternal Control of PIN1 Is Required for Female Gametophyte Development in Arabidopsis

Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.     

Somatic small RNA pathways promote the mitotic events of megagametogenesis during female reproductive development in Arabidopsis

Female gamete development in Arabidopsis ovules comprises two phases. During megasporogenesis, a somatic ovule cell differentiates into a megaspore mother cell and undergoes meiosis to produce four haploid megaspores, three of which degrade. The surviving functional megaspore participates in megagametogenesis, undergoing syncytial mitosis and cellular differentiation to produce a multicellular female gametophyte containing the egg and central cell, progenitors of the embryo and endosperm of the seed. The transition between megasporogenesis and megagametogenesis is poorly characterised, partly owing to the inaccessibility of reproductive cells within the ovule. Here, laser capture microdissection was used to identify genes expressed in and/or around developing megaspores during the transition to megagametogenesis. ARGONAUTE5 (AGO5), a putative effector of small RNA (sRNA) silencing pathways, was found to be expressed around reproductive cells during megasporogenesis, and a novel semi-dominant ago5-4 insertion allele showed defects in the initiation of megagametogenesis. Expression of a viral RNAi suppressor, P1/Hc-Pro, driven by the WUSCHEL and AGO5 promoters in somatic cells flanking the megaspores resulted in a similar phenotype. This indicates that sRNA-dependent pathways acting in somatic ovule tissues promote the initiation of megagametogenesis in the functional megaspore. Notably, these pathways are independent of AGO9, which functions in somatic epidermal ovule cells to inhibit the formation of multiple megaspore-like cells. Therefore, one somatic sRNA pathway involving AGO9 restricts reproductive development to the functional megaspore and a second pathway, inhibited by ago5-4 and P1/Hc-Pro, promotes megagametogenesis.     

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.     

Identification of genes expressed in the angiosperm female gametophyte

Until recently, identification of gene regulatory networks controlling the development of the angiosperm female gametophyte has presented a significant challenge to the plant biology community. The angiosperm female gametophyte is fairly inaccessible because it is a highly reduced structure relative to the sporophyte and is embedded within multiple layers of the sporophytic tissue of the ovule. Moreover, although mutations affecting the female gametophyte can be readily isolated, their analysis can be difficult because most affect genes involved in basic cellular processes that are also required in the diploid sporophyte. In recent years, expression-based approaches in multiple species have begun to uncover gene sets expressed in specific female gametophyte cells as a means of identifying regulatory networks controlling cell differentiation in the female gametophyte. Here, recent efforts to identify and analyse gene expression programmes in the Arabidopsis female gametophyte are reviewed.     

Maternal Gametophyte Effects on Seed Development in Maize

Flowering plants, like placental mammals, have an extensive maternal contribution toward progeny development. Plants are distinguished from animals by a genetically active haploid phase of growth and development between meiosis and fertilization, called the gametophyte. Flowering plants are further distinguished by the process of double fertilization that produces sister progeny, the endosperm and the embryo, of the seed. Because of this, there is substantial gene expression in the female gametophyte that contributes to the regulation of growth and development of the seed. A primary function of the endosperm is to provide growth support to its sister embryo. Several mutations in Zea mays subsp. mays have been identified that affect the contribution of the mother gametophyte to the seed. The majority affect both the endosperm and the embryo, although some embryo-specific effects have been observed. Many alter the pattern of expression of a marker for the basal endosperm transfer layer, a tissue that transports nutrients from the mother plant to the developing seed. Many of them cause abnormal development of the female gametophyte prior to fertilization, revealing potential cellular mechanisms of maternal control of seed development. These effects include reduced central cell size, abnormal architecture of the central cell, abnormal numbers and morphology of the antipodal cells, and abnormal egg cell morphology. These mutants provide insight into the logic of seed development, including necessary features of the gametes and supporting cells prior to fertilization, and set up future studies on the mechanisms regulating maternal contributions to the seed.     

POD1 regulates pollen tube guidance in response to micropylar female signaling and acts in early embryo patterning in Arabidopsis

The pollen tube germinates from pollen and, during its migration, it perceives and responds to guidance cues from maternal tissue and from the female gametophyte. The putative female cues have recently been identified, but how the pollen tube responds to these signals remains to be unveiled. In a genetic screen for male determinants of the pollen tube response, we identified the pollen defective in guidance1 (pod1) mutant, in which the pollen tubes fail to target the female gametophyte. POD1 encodes a conserved protein of unknown function and is essential for positioning and orienting the cell division plane during early embryo development. Here, we demonstrate that POD1 is an endoplasmic reticulum (ER) luminal protein involved in ER protein retention. Further analysis shows that POD1 interacts with the Ca(2+) binding ER chaperone CALRETICULIN3 (CRT3), a protein in charge of folding of membrane receptors. We propose that POD1 modulates the activity of CRT3 or other ER resident factors to control the folding of proteins, such as membrane proteins in the ER. By this mechanism, POD1 may regulate the pollen tube response to signals from the female tissues during pollen tube guidance and early embryo patterning in Arabidopsis thaliana.     

Haplo-insufficiency of MPK3 in MPK6 mutant background uncovers a novel function of these two MAPKs in Arabidopsis ovule development

The plant life cycle includes diploid sporophytic and haploid gametophytic generations. Female gametophytes (embryo sacs) in higher plants are embedded in specialized sporophytic structures (ovules). Here, we report that two closely related mitogen-activated protein kinases in Arabidopsis thaliana, MPK3 and MPK6, share a novel function in ovule development: in the MPK6 mutant background, MPK3 is haplo-insufficient, giving female sterility when heterozygous. By contrast, in the MPK3 mutant background, MPK6 does not show haplo-insufficiency. Using wounding treatment, we discovered gene dosage-dependent activation of MPK3 and MPK6. In addition, MPK6 activation is enhanced when MPK3 is null, which may help explain why mpk3(-/-) mpk6(+/-) plants are fertile. Genetic analysis revealed that the female sterility of mpk3(+/-) mpk6(-/-) plants is a sporophytic effect. In mpk3(+/-) mpk6(-/-) mutant plants, megasporogenesis and megagametogenesis are normal and the female gametophyte identity is correctly established. Further analysis demonstrates that the mpk3(+/-) mpk6(-/-) ovules have abnormal integument development with arrested cell divisions at later stages. The mutant integuments fail to accommodate the developing embryo sac, resulting in the embryo sacs being physically restricted and female reproductive failure. Our results highlight an essential function of MPK3 and MPK6 in promoting cell division in the integument specifically during ovule development.     

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.     

Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart. Dev. Genet. 22:288–299, 1998. © 1998 Wiley-Liss, Inc.