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1.  Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis 
eLife  null;4:e06587.
The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor.
eLife digest
Plants respond to changes in their environment by altering how they grow and when they reproduce. A protein called FERONIA is found in most types of cells and regulates many of the processes that drive these responses, such as cell growth and communication between male and female cells. FERONIA sits in the membrane that surrounds the cell, where it can detect molecules in the cell wall and from outside the cell, and send signals to locations within the cell. However, it is not clear how FERONIA is able to specifically regulate different processes to produce the right response in a particular cell at a particular time.
A family of proteins called glycosylphosphatidylinositol-anchored proteins (GPI-APs for short) play important roles in plants, animals, and other eukaryotic organisms. Li et al. studied FERONIA and two closely related GPI-APs called LLG1—which is produced in seedlings, and LORELEI, which is only found in female sex cells. The experiments show that plants missing either LLG1 or FERONIA had similar defects in growth and in how they respond to plant hormones. Plants missing LORELEI had similar defects in their ability to reproduce as the plants missing FERONIA. This suggests that FERONIA works with either LLG1 or LORELEI to regulate similar processes in different situations.
Li et al. found that FERONIA binds to LLG1 in a compartment within the cell called the endoplasmic reticulum—where proteins are assembled—before both proteins are moved together to the cell membrane. In the absence of LLG1, FERONIA fails to reach the cell membrane, and a large amount of FERONIA remains trapped in the endoplasmic reticulum. Therefore, LLG1 acts as a ‘chaperone’ that delivers FERONIA to the membrane where it is required to regulate plant growth. Li et al. found that LORELEI also interacts with FERONIA. Both LLG1 and LORELEI bind to the same region of FERONIA, which is on the outer surface of the cell membrane.
These findings show that FERONIA is able to perform different roles in cells by teaming up with different members of the GPI-AP family of proteins. The next challenges will be to find out if, and how, LLG1 and LORELEI affect the ability of FERONIA to respond to signals from the cell wall and outside the cell.
PMCID: PMC4458842  PMID: 26052747
malectin domain-containing receptor kinases; FERONIA and LORELEI/LLG1 as coreceptors; receptor kinase and GPI-AP as functional partners; plant Rho GTPase signaling; RALF; Arabidopsis
2.  Pollination triggers female gametophyte development in immature Nicotiana tabacum flowers 
In Nicotiana tabacum, female gametophytes are not fully developed at anthesis, but flower buds pollinated 12 h before anthesis produce mature embryo sacs. We investigated several pollination-associated parameters in N. tabacum flower buds to determine the developmental timing of important events in preparation for successful fertilization. First, we performed hand pollinations in flowers from stages 4 to 11 to study at which developmental stage pollination would produce fruits. A Peroxtesmo test was performed to correlate peroxidase activity on the stigma surface, indicative of stigma receptivity, with fruit set. Pollen tube growth and female gametophyte development were microscopically analyzed in pistils of different developmental stages. Fruits were obtained only after pollinations of flower buds at late stage 7 and older; fruit weight and seed germination capacity increased as the developmental stage of the pollinated flower approached anthesis. Despite positive peroxidase activity and pollen tube growth, pistils at stages 5 and 6 were unable to produce fruits. At late stage 7, female gametophytes were undergoing first mitotic division. After 24 h, female gametophytes of unpollinated pistils were still in the end of the first division, whereas those of pollinated pistils showed egg cells. RT-qPCR assay showed that the expression of the NtEC1 gene, a marker of egg cell development, is considerably higher in pollinated late stage 7 ovaries compared with unpollinated ovaries. To test whether ethylene is the signal eliciting female gametophyte maturation, the expression of ACC synthase was examined in unpollinated and pollinated stage 6 and late stage 7 stigmas/styles. Pollination induced NtACS expression in stage 6 pistils, which are unable to produce fruits. Our results show that pollination is a stimulus capable of triggering female gametophyte development in immature tobacco flowers and suggests the existence of a yet undefined signal sensed by the pistil.
PMCID: PMC4510347  PMID: 26257764
stigma receptivity; pollen tube growth; pollination signal; female gametophyte development; fruit weight; seed germination capacity
3.  Genome-Wide Expression Profiling of the Arabidopsis Female Gametophyte Identifies Families of Small, Secreted Proteins 
PLoS Genetics  2007;3(10):e171.
The female gametophyte of flowering plants, the embryo sac, develops within the diploid (sporophytic) tissue of the ovule. While embryo sac–expressed genes are known to be required at multiple stages of the fertilization process, the set of embryo sac–expressed genes has remained poorly defined. In particular, the set of genes responsible for mediating intracellular communication between the embryo sac and the male gametophyte, the pollen grain, is unknown. We used high-throughput cDNA sequencing and whole-genome tiling arrays to compare gene expression in wild-type ovules to that in dif1 ovules, which entirely lack embryo sacs, and myb98 ovules, which are impaired in pollen tube attraction. We identified nearly 400 genes that are downregulated in dif1 ovules. Seventy-eight percent of these embryo sac–dependent genes were predicted to encode for secreted proteins, and 60% belonged to multigenic families. Our results define a large number of candidate extracellular signaling molecules that may act during embryo sac development or fertilization; less than half of these are represented on the widely used ATH1 expression array. In particular, we found that 37 out of 40 genes encoding Domain of Unknown Function 784 (DUF784) domains require the synergid-specific transcription factor MYB98 for expression. Several DUF784 genes were transcribed in synergid cells of the embryo sac, implicating the DUF784 gene family in mediating late stages of embryo sac development or interactions with pollen tubes. The coexpression of highly similar proteins suggests a high degree of functional redundancy among embryo sac genes.
Author Summary
During the sexual reproduction of flowering plants, a pollen tube delivers sperm cells to a specialized group of cells known as the embryo sac, which contains the egg cell. It is known that embryo sacs are active participants in guiding the growth of pollen tubes, in facilitating fertilization, and in initiating seed development. However, the genes responsible for the complex biology of embryo sacs are poorly understood. The authors use two recently developed technologies, whole-genome tiling microarrays and high-throughput cDNA sequencing, to identify hundreds of genes expressed in embryo sacs of Arabidopsis thaliana. Most embryo sac–dependent genes have no known function, and include entire families of related genes that are only expressed in embryo sacs. Furthermore, most embryo sac–dependent genes encode small proteins that are potentially secreted from their cells of origin, suggesting that they may act as intracellular signals or to modify the extracellular matrix during fertilization or embryo sac development. These results illustrate the extent to which our understanding of plant sexual reproduction is limited and identifies hundreds of candidate genes for future studies investigating the molecular biology of the embryo sac.
PMCID: PMC2014789  PMID: 17937500
4.  The beginning of a seed: regulatory mechanisms of double fertilization 
The launch of seed development in flowering plants (angiosperms) is initiated by the process of double fertilization: two male gametes (sperm cells) fuse with two female gametes (egg and central cell) to form the precursor cells of the two major seed components, the embryo and endosperm, respectively. The immobile sperm cells are delivered by the pollen tube toward the ovule harboring the female gametophyte by species-specific pollen tube guidance and attraction mechanisms. After pollen tube burst inside the female gametophyte, the two sperm cells fuse with the egg and central cell initiating seed development. The fertilized central cell forms the endosperm while the fertilized egg cell, the zygote, will form the actual embryo and suspensor. The latter structure connects the embryo with the sporophytic maternal tissues of the developing seed. The underlying mechanisms of double fertilization are tightly regulated to ensure delivery of functional sperm cells and the formation of both, a functional zygote and endosperm. In this review we will discuss the current state of knowledge about the processes of directed pollen tube growth and its communication with the synergid cells resulting in pollen tube burst, the interaction of the four gametes leading to cell fusion and finally discuss mechanisms how flowering plants prevent multiple sperm cell entry (polyspermy) to maximize their reproductive success.
PMCID: PMC4160995  PMID: 25309552
pollen tube; ovule; gamete interaction; cell fusion; signaling; fertilization; polyspermy
5.  Cytokinin receptors in sporophytes are essential for male and female functions in Arabidopsis thaliana 
Plant Signaling & Behavior  2011;6(1):66-71.
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.
PMCID: PMC3122008  PMID: 21301212
cytokinin; cytokinin receptor; female gametophyte; male gametophyte; stigma
6.  The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development 
Journal of Experimental Botany  2015;66(7):1833-1843.
This study reveals the ScFRK1 MAP kinase kinase kinase as a novel player in male and female gametophyte development, ultimately affecting pollen tube guidance and gametophyte to sporophyte communication.
The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development.
PMCID: PMC4378624  PMID: 25576576
Embryo sac development; gametophyte to sporophyte communication; MAPKKK; megagametogenesis; microgametogenesis; pollen tube guidance; seed and fruit development; Solanaceae.
7.  TURAN and EVAN Mediate Pollen Tube Reception in Arabidopsis Synergids through Protein Glycosylation 
PLoS Biology  2015;13(4):e1002139.
Pollen tube (PT) reception in flowering plants describes the crosstalk between the male and female gametophytes upon PT arrival at the synergid cells of the ovule. It leads to PT growth arrest, rupture, and sperm cell release, and is thus essential to ensure double fertilization. Here, we describe TURAN (TUN) and EVAN (EVN), two novel members of the PT reception pathway that is mediated by the FERONIA (FER) receptor-like kinase (RLK). Like fer, mutations in these two genes lead to PT overgrowth inside the female gametophyte (FG) without PT rupture. Mapping by next-generation sequencing, cytological analysis of reporter genes, and biochemical assays of glycoproteins in RNAi knockdown mutants revealed both genes to be involved in protein N-glycosylation in the endoplasmic reticulum (ER). TUN encodes a uridine diphosphate (UDP)-glycosyltransferase superfamily protein and EVN a dolichol kinase. In addition to their common role during PT reception in the synergids, both genes have distinct functions in the pollen: whereas EVN is essential for pollen development, TUN is required for PT growth and integrity by affecting the stability of the pollen-specific FER homologs ANXUR1 (ANX1) and ANX2. ANX1- and ANX2-YFP reporters are not expressed in tun pollen grains, but ANX1-YFP is degraded via the ER-associated degradation (ERAD) pathway, likely underlying the anx1/2-like premature PT rupture phenotype of tun mutants. Thus, as in animal sperm–egg interactions, protein glycosylation is essential for the interaction between the female and male gametophytes during PT reception to ensure fertilization and successful reproduction.
Protein glycosylation is essential for gametophyte interactions between the male pollen tube and the female ovule in plants, reminiscent of gamete interactions during fertilization in mammals.
Author Summary
In flowering plants, gametes are produced by the haploid, multicellular male (pollen), and female (embryo sac) gametophytes, which develop within the reproductive organs of the flower. Successful fertilization depends on delivery of the sperm cells to the embryo sac, which is embedded in the ovule, by the pollen tube. Upon arrival of the pollen tube at the opening of the ovule, crosstalk between male and female gametophytes, known as pollen tube reception, ensues; the pollen tube slows or stops its growth, then resumes rapid growth, and finally bursts to release the sperm cells and effect double fertilization. Although several members of the pollen tube reception pathway, including the receptor-like kinase FERONIA, have been identified, the molecular mechanisms underlying this communication process remain unclear. Here, we show that protein N-glycosylation is required for normal pollen tube reception. A mutant screen identified two genes, TURAN and EVAN, which are involved in protein N-glycosylation in the endoplasmic reticulum. Both genes act in the FERONIA-mediated pollen tube reception pathway, which is impaired in these mutants. Thus, in plants, a “dual recognition system,” involving interactions between both protein and glycosyl residues on the surface of male and female gametophytes, appears to be required for successful pollen tube reception, conceptually similar to sperm–egg interactions in mammals, for which N-glycosylation of cell surface proteins also plays an important role.
PMCID: PMC4412406  PMID: 25919390
8.  The Female Gametophyte 
The angiosperm female gametophyte is critical for plant reproduction. It contains the egg cell and central cell that become fertilized and give rise to the embryo and endosperm of the seed, respectively. Female gametophyte development begins early in ovule development with the formation of a diploid megaspore mother cell that undergoes meiosis. One resulting haploid megaspore then develops into the female gametophyte. Genetic and epigenetic processes mediate specification of megaspore mother cell identity and limit megaspore mother cell formation to a single cell per ovule. Auxin gradients influence female gametophyte polarity and a battery of transcription factors mediate female gametophyte cell specification and differentiation. The mature female gametophyte secretes peptides that guide the pollen tube to the embryo sac and contains protein complexes that prevent seed development before fertilization. Post-fertilization, the female gametophyte influences seed development through maternal-effect genes and by regulating parental contributions. Female gametophytes can form by an asexual process called gametophytic apomixis, which involves formation of a diploid female gametophyte and fertilization-independent development of the egg into the embryo. These functions collectively underscore the important role of the female gametophyte in seed and food production.
PMCID: PMC3268550  PMID: 22303279
9.  Sporophytic control of pollen tube growth and guidance in maize 
Journal of Experimental Botany  2009;61(3):673-682.
Pollen tube germination, growth, and guidance (progamic phase) culminating in sperm discharge is a multi-stage process including complex interactions between the male gametophyte as well as sporophytic tissues and the female gametophyte (embryo sac), respectively. Inter- and intra-specific crossing barriers in maize and Tripsacum have been studied and a precise description of progamic pollen tube development in maize is reported here. It was found that pollen germination and initial tube growth are rather unspecific, but an early, first crossing barrier was detected before arrival at the transmitting tract. Pollination of maize silks with Tripsacum pollen and incompatible pollination of Ga1s/Ga1s-maize silks with ga1-maize pollen revealed another two incompatibility barriers, namely transmitting tract mistargeting and insufficient growth support. Attraction and growth support by the transmitting tract seem to play key roles for progamic pollen tube growth. After leaving transmitting tracts, pollen tubes have to navigate across the ovule in the ovular cavity. Pollination of an embryo sac-less maize RNAi-line allowed the role of the female gametophyte for pollen tube guidance to be determined in maize. It was found that female gametophyte controlled guidance is restricted to a small region around the micropyle, approximately 50–100 μm in diameter. This area is comparable to the area of influence of previously described ZmEA1-based short-range female gametophyte signalling. In conclusion, the progamic phase is almost completely under sporophytic control in maize.
PMCID: PMC2814102  PMID: 19926683
Female gametophyte; maize; pollen tube guidance; prezygotic barriers; transmitting tract; Tripsacum
10.  Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey 
Plant Signaling & Behavior  2013;8(12):e27377.
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.
PMCID: PMC4091341  PMID: 24384993
CRP; EC1; double fertilization; gamete fusion; polytubey; sperm activation
11.  Using maize as a model to study pollen tube growth and guidance, cross-incompatibility and sperm delivery in grasses 
Annals of Botany  2011;108(4):727-737.
In contrast to animals and lower plants such as mosses and ferns, sperm cells of flowering plants (angiosperms) are immobile and require transportation to the female gametes via the vegetative pollen tube cell to achieve double fertilization. The path of the pollen tube towards the female gametophyte (embryo sac) has been intensively studied in many intra- and interspecific crossing experiments with the aim of increasing the gene pool of crop plants for greater yield, improved biotic and abiotic stress resistance, and for introducing new agronomic traits. Many attempts to hybridize different species or genotypes failed due to the difficulty for the pollen tubes in reaching the female gametophyte. Detailed studies showed that these processes are controlled by various self-incompatible (intraspecific) and cross-incompatible (interspecific) hybridization mechanisms.
Understanding the molecular mechanisms of crossing barriers is therefore of great interest in plant reproduction, evolution and breeding research. In particular, pre-zygotic hybridization barriers related to pollen tube germination, growth, guidance and sperm delivery, which are considered the major hybridization controls in nature and thus also contribute to species isolation and speciation, have been intensively investigated. Despite this general interest, surprisingly little is known about these processes in the most important agronomic plant family, the Gramineae, Poaceae or grasses. Small polymorphic proteins and their receptors, degradation of sterility locus proteins and general compounds such as calcium, γ-aminobutyric acid or nitric oxide have been shown to be involved in progamic pollen germination, adhesion, tube growth and guidance, as well as sperm release. Most advances have been made in the Brassicaceae, Papaveraceae, Linderniaceae and Solanaceae families including their well-understood self-incompatibility (SI) systems. Grass species evolved similar mechanisms to control the penetration and growth of self-pollen to promote intraspecific outcrossing and to prevent fertilization by alien sperm cells. However, in the Poaceae, the underlying molecular mechanisms are still largely unknown.
We propose to develop maize (Zea mays) as a model to investigate the above-described processes to understand the associated intra- and interspecific crossing barriers in grasses. Many genetic, cellular and biotechnological tools including the completion of a reference genome (inbred line B73) have been established in the last decade and many more maize inbred genomes are expected to be available soon. Moreover, a cellular marker line database as well as large transposon insertion collections and improved Agrobacterium transformation protocols are now available. Additionally, the processes described above are well studied at the morphological level and a number of mutants have been described already, awaiting disclosure of the relevant genes. The identification of the first key players in pollen tube growth, guidance and burst show maize to be an excellent grass model to investigate these processes in more detail. Here we provide an overview of our current understanding of these processes in Poaceae with a focus on maize, and also include relevant discoveries in eudicot model species.
PMCID: PMC3170146  PMID: 21345919
Maize; male germline; sperm cell; interspecific crosses; self- and cross-incompatibility; pollen tube growth and guidance; fertilization; reproductive isolation
12.  Genome-Wide Transcript Profiling of Endosperm without Paternal Contribution Identifies Parent-of-Origin–Dependent Regulation of AGAMOUS-LIKE36 
PLoS Genetics  2011;7(2):e1001303.
Seed development in angiosperms is dependent on the interplay among different transcriptional programs operating in the embryo, the endosperm, and the maternally-derived seed coat. In angiosperms, the embryo and the endosperm are products of double fertilization during which the two pollen sperm cells fuse with the egg cell and the central cell of the female gametophyte. In Arabidopsis, analyses of mutants in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CKDA;1) have revealed the importance of a paternal genome for the effective development of the endosperm and ultimately the seed. Here we have exploited cdka;1 fertilization as a novel tool for the identification of seed regulators and factors involved in parent-of-origin–specific regulation during seed development. We have generated genome-wide transcription profiles of cdka;1 fertilized seeds and identified approximately 600 genes that are downregulated in the absence of a paternal genome. Among those, AGAMOUS-LIKE (AGL) genes encoding Type-I MADS-box transcription factors were significantly overrepresented. Here, AGL36 was chosen for an in-depth study and shown to be imprinted. We demonstrate that AGL36 parent-of-origin–dependent expression is controlled by the activity of METHYLTRANSFERASE1 (MET1) maintenance DNA methyltransferase and DEMETER (DME) DNA glycosylase. Interestingly, our data also show that the active maternal allele of AGL36 is regulated throughout endosperm development by components of the FIS Polycomb Repressive Complex 2 (PRC2), revealing a new type of dual epigenetic regulation in seeds.
Author Summary
Seeds of flowering plants consist of three different organisms that develop in parallel. In contrast to animals, a double fertilization event takes place in plants, producing two fertilization products, the embryo and the endosperm. Imprinting, the parent-of-origin–specific expression of genes, typically takes place in the mammalian placenta and in the plant endosperm. A prevailing hypothesis predicts that a parental tug-of-war on the allocation of available recourses to the developing progeny has led to the evolution of imprinting systems where genes expressed from the mother dampen growth whereas genes expressed from the father are growth enhancers. The number of imprinted genes identified in plants is low compared to mammals, and this precludes the elucidation of the epigenetic mechanisms responsible for this specialized expression system. Here, we have used genome-wide transcript profiling of endosperm without paternal contribution to identify seed regulators and, among these, imprinted genes. We identified a cluster of downregulated MADS-box transcription factors, including AGL36, that was subsequently shown to be imprinted by an epigenetic mechanism involving the DNA methylase MET1 and the glycosylase DME. In addition, the expression of the active AGL36 allele was dampened by the FIS Polycomb Repressive Complex, identifying a novel mode of regulation of imprinted genes.
PMCID: PMC3040660  PMID: 21379330
13.  The Peroxin Loss-of-Function Mutation abstinence by mutual consent Disrupts Recognition Between Male and Female Gametophytes1 
Current biology : CB  2007;18(1):63-68.
In eukaryotes, fertilization relies on complex and specialized mechanisms that achieve the precise delivery of the male gamete to the female gamete and their subsequent union [1–4]. In flowering plants, the haploid male gametophyte or pollen tube (PT) [5] carries two non-motile sperm cells to the female gametophyte (FG) or embryo sac [6] during a long assisted journey through the maternal tissues [7–10]. In Arabidopsis, typically one PT reaches one of the two synergids of the FG (Figure 1A) where it terminates its growth and delivers the sperm cells, a poorly understood process called pollen tube reception. Here, we report the isolation and characterization of the Arabidopsis mutant abstinence by mutual consent. Interestingly, pollen tube reception is impaired only when an amc pollen tube reaches an amc female gametophyte resulting in pollen tube overgrowth and completely preventing sperm discharge and the development of homozygous mutants. Moreover, we show that AMC is strongly and transiently expressed in both male and female gametophytes during fertilization and that AMC functions in gametophytes as a peroxin essential for protein import into peroxisomes. These findings show that peroxisomes play an unexpected key role in gametophyte recognition and implicate a diffusible signal emanating from either gametophytes that is required for pollen tube discharge.
PMCID: PMC2278027  PMID: 18160292
14.  Identification of transcription-factor genes expressed in the Arabidopsis female gametophyte 
BMC Plant Biology  2010;10:110.
In flowering plants, the female gametophyte is typically a seven-celled structure with four cell types: the egg cell, the central cell, the synergid cells, and the antipodal cells. These cells perform essential functions required for double fertilization and early seed development. Differentiation of these distinct cell types likely involves coordinated changes in gene expression regulated by transcription factors. Therefore, understanding female gametophyte cell differentiation and function will require dissection of the gene regulatory networks operating in each of the cell types. These efforts have been hampered because few transcription factor genes expressed in the female gametophyte have been identified. To identify such genes, we undertook a large-scale differential expression screen followed by promoter-fusion analysis to detect transcription-factor genes transcribed in the Arabidopsis female gametophyte.
Using quantitative reverse-transcriptase PCR, we analyzed 1,482 Arabidopsis transcription-factor genes and identified 26 genes exhibiting reduced mRNA levels in determinate infertile 1 mutant ovaries, which lack female gametophytes, relative to ovaries containing female gametophytes. Spatial patterns of gene transcription within the mature female gametophyte were identified for 17 transcription-factor genes using promoter-fusion analysis. Of these, ten genes were predominantly expressed in a single cell type of the female gametophyte including the egg cell, central cell and the antipodal cells whereas the remaining seven genes were expressed in two or more cell types. After fertilization, 12 genes were transcriptionally active in the developing embryo and/or endosperm.
We have shown that our quantitative reverse-transcriptase PCR differential-expression screen is sufficiently sensitive to detect transcription-factor genes transcribed in the female gametophyte. Most of the genes identified in this study have not been reported previously as being expressed in the female gametophyte. Therefore, they might represent novel regulators and provide entry points for reverse genetic and molecular approaches to uncover the gene regulatory networks underlying female gametophyte development.
PMCID: PMC3236301  PMID: 20550711
15.  Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1 
PLoS Biology  2010;8(6):e1000388.
Species-preferential osmotic pollen tube burst and sperm discharge in maize involve induced opening of the pollen tube-expressed potassium channel KZM1 by the egg apparatus-derived defensin-like protein ZmES4.
In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i.e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4 (Zea mays embryo sac) from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K+-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K+ channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K+ influx. We further suggest that K+ influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization barrier and may be a component of reproductive isolation in plants.
Author Summary
Sperm cells of animals and lower plants are mobile and can swim to the oocyte or egg cell. In contrast, flowering plants generate immobile sperm encased in a pollen coat to protect them from drying out and are transported via the pollen tube cell towards the egg apparatus to achieve double fertilization. Upon arrival the pollen tube tip bursts to deliver two sperm cells, one fusing with the egg cell to generate the embryo and the other fusing with the central cell to generate the endosperm. Here, we report the mechanisms leading to pollen tube burst and sperm discharge in maize. We found that before fertilization the defensin-like protein ZmES1-4 is stored in the secretory zone of the egg apparatus cells and that pollen tubes cannot discharge sperm in ZmES1-4 knock-down plants. Application of chemically synthesized ZmES4 leads to pollen tube burst within seconds in maize, but not in other plant species, suggesting this mechanism may be species specific. Finally, we identified the pollen tube-expressed potassium channel KZM1 as a target of ZmES4, which opens after ZmES4 treatment and probably leads to K+ influx and sperm release after osmotic burst.
PMCID: PMC2879413  PMID: 20532241
16.  YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis 
BMC Plant Biology  2010;10:169.
In flowering plants, gametogenesis generates multicellular male and female gametophytes. In the model system Arabidopsis, the male gametophyte or pollen grain contains two sperm cells and a vegetative cell. The female gametophyte or embryo sac contains seven cells, namely one egg, two synergids, one central cell and three antipodal cells. Double fertilization of the central cell and egg produces respectively a triploid endosperm and a diploid zygote that develops further into an embryo. The genetic control of the early embryo patterning, especially the initiation of the first zygotic division and the positioning of the cell plate, is largely unknown.
Here we report the characterization of a mutation, yaozhe (yao), that causes zygote arrest and misplacement of cell plate of the zygote, leading to early embryo lethality. In addition, gametophyte development is partially impaired. A small portion of the mutant embryo sacs are arrested at four-nucleate stage with aberrant nuclear positioning. Furthermore, the competence of male gametophytes is also compromised. YAO encodes a nucleolar protein with seven WD-repeats. Its homologues in human and yeast have been shown to be components of the U3 snoRNP complex and function in 18S rRNA processing. YAO is expressed ubiquitously, with high level of expression in tissues under active cell divisions, including embryo sacs, pollen, embryos, endosperms and root tips.
Phenotypic analysis indicated that YAO is required for the correct positioning of the first zygotic division plane and plays a critical role in gametogenesis in Arabidopsis. Since YAO is a nucleolar protein and its counterparts in yeast and human are components of the U3 snoRNP complex, we therefore postulate that YAO is most likely involved in rRNA processing in plants as well.
PMCID: PMC3095302  PMID: 20699009
17.  Sulfinylated Azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils 
Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultrahigh resolution ESI FT-ICR and MS/MS techniques to accurately determine the mass (202.126 daltons) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C10H19NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N-Methanesulfinyl 1- and 2-azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 µM stimulated ~50% germination) and elicit accession-specific response. Although N-Methanesulfinyl 2-azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin-like molecules to ensure rapid fertilization by the appropriate pollen.
PMCID: PMC3225508  PMID: 21801250
Pollen; pistil; germination; stimulant; chemical biology; functional mimic
18.  Arabidopsis thaliana mitogen-activated protein kinase 6 is involved in seed formation and modulation of primary and lateral root development 
Journal of Experimental Botany  2013;65(1):169-183.
Arabidopsis MAP kinases are considered to have redundant functions. However, through a detailed phenotypic analysis, we demonstrated that MPK6 loss-of- function cause severe defects in embryo development, which are closed related with alterations in post-germination root development
Mitogen-activated protein kinase (MAPKs) cascades are signal transduction modules highly conserved in all eukaryotes regulating various aspects of plant biology, including stress responses and developmental programmes. In this study, we characterized the role of MAPK 6 (MPK6) in Arabidopsis embryo development and in post-embryonic root system architecture. We found that the mpk6 mutation caused altered embryo development giving rise to three seed phenotypes that, post-germination, correlated with alterations in root architecture. In the smaller seed class, mutant seedlings failed to develop the primary root, possibly as a result of an earlier defect in the division of the hypophysis cell during embryo development, but they had the capacity to develop adventitious roots to complete their life cycle. In the larger class, the MPK6 loss of function did not cause any evident alteration in seed morphology, but the embryo and the mature seed were bigger than the wild type. Seedlings developed from these bigger seeds were characterized by a primary root longer than that of the wild type, accompanied by significantly increased lateral root initiation and more and longer root hairs. Apparently, the increment in primary root growth resulted from an enhanced cell production and cell elongation. Our data demonstrated that MPK6 plays an important role during embryo development and acts as a repressor of primary and lateral root development.
PMCID: PMC3883294  PMID: 24218326
Arabidopsis; embryo development; MAP kinases; MPK6; plant signalling; root development.
19.  CENH3-GFP: a visual marker for gametophytic and somatic ploidy determination in Arabidopsis thaliana 
BMC Plant Biology  2016;16:1.
The in vivo determination of the cell-specific chromosome number provides a valuable tool in several aspects of plant research. However, current techniques to determine the endosystemic ploidy level do not allow non-destructive, cell-specific chromosome quantification. Particularly in the gametophytic cell lineages, which are physically encapsulated in the reproductive organ structures, direct in vivo ploidy determination has been proven very challenging. Using Arabidopsis thaliana as a model, we here assess the applicability of recombinant CENH3-GFP reporters for the labeling of the cell’s chromocenters and for the monitoring of the gametophytic and somatic chromosome number in vivo.
By modulating expression of a CENH3-GFP reporter cassette using different promoters, we isolated two reporter lines that allow for a clear and highly specific labeling of centromeric chromosome regions in somatic and gametophytic cells respectively. Using polyploid plant series and reproductive mutants, we demonstrate that the pWOX2-CENH3-GFP recombinant fusion protein allows for the determination of the gametophytic chromosome number in both male and female gametophytic cells, and additionally labels centromeric regions in early embryo development. Somatic centromere labeling through p35S-CENH3-GFP shows a maximum of ten centromeric dots in young dividing tissues, reflecting the diploid chromosome number (2x = 10), and reveals a progressive decrease in GFP foci frequency throughout plant development. Moreover, using chemical and genetic induction of endomitosis, we demonstrate that CENH3-mediated chromosome labeling provides an easy and valuable tool for the detection and characterization of endomitotic polyploidization events.
This study demonstrates that the introgression of the pWOX2-CENH3-GFP reporter construct in Arabidopsis thaliana provides an easy and reliable methodology for determining the chromosome number in developing male and female gametes, and during early embryo development. Somatically expressed CENH3-GFP reporters, on the other hand, constitute a valuable tool to quickly determine the basic somatic ploidy level in young seedlings at the individual cell level and to detect and to quantify endomitotic polyploidization events in a non-destructive, microscopy-based manner.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-015-0700-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4700667  PMID: 26728271
CENH3; Centromere; Arabidopsis; Ploidy analysis; Meiosis; Endomitosis
20.  Discovery of novel transcripts and gametophytic functions via RNA-seq analysis of maize gametophytic transcriptomes 
Genome Biology  2014;15(7):414.
Plant gametophytes play central roles in sexual reproduction. A hallmark of the plant life cycle is that gene expression is required in the haploid gametophytes. Consequently, many mutant phenotypes are expressed in this phase.
We perform a quantitative RNA-seq analysis of embryo sacs, comparator ovules with the embryo sacs removed, mature pollen, and seedlings to assist the identification of gametophyte functions in maize. Expression levels were determined for annotated genes in both gametophytes, and novel transcripts were identified from de novo assembly of RNA-seq reads. Transposon-related transcripts are present in high levels in both gametophytes, suggesting a connection between gamete production and transposon expression in maize not previously identified in any female gametophytes. Two classes of small signaling proteins and several transcription factor gene families are enriched in gametophyte transcriptomes. Expression patterns of maize genes with duplicates in subgenome 1 and subgenome 2 indicate that pollen-expressed genes in subgenome 2 are retained at a higher rate than subgenome 2 genes with other expression patterns. Analysis of available insertion mutant collections shows a statistically significant deficit in insertions in gametophyte-expressed genes.
This analysis, the first RNA-seq study to compare both gametophytes in a monocot, identifies maize gametophyte functions, gametophyte expression of transposon-related sequences, and unannotated, novel transcripts. Reduced recovery of mutations in gametophyte-expressed genes is supporting evidence for their function in the gametophytes. Expression patterns of extant, duplicated maize genes reveals that selective pressures based on male gametophytic function have likely had a disproportionate effect on plant genomes.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0414-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4309534  PMID: 25084966
21.  The Arabidopsis thaliana F-Box Protein FBL17 Is Essential for Progression through the Second Mitosis during Pollen Development 
PLoS ONE  2009;4(3):e4780.
In fungi and metazoans, the SCF-type Ubiquitin protein ligases (E3s) play a critical role in cell cycle regulation by degrading negative regulators, such as cell cycle-dependent kinase inhibitors (CKIs) at the G1-to-S-phase checkpoint. Here we report that FBL17, an Arabidopsis thaliana F-box protein, is involved in cell cycle regulation during male gametogenesis. FBL17 expression is strongly enhanced in plants co-expressing E2Fa and DPa, transcription factors that promote S-phase entry. FBL17 loss-of-function mutants fail to undergo pollen mitosis II, which generates the two sperm cells in mature A. thaliana pollen. Nonetheless, the single sperm cell-like cell in fbl17 mutants is functional but will exclusively fertilize the egg cell of the female gametophyte, giving rise to an embryo that will later abort, most likely due to the lack of functional endosperm. Seed abortion can, however, be overcome by mutations in FIE, a component of the Polycomb group complex, overall resembling loss-of-function mutations in the A. thaliana cyclin-dependent kinase CDKA;1. Finally we identified ASK11, as an SKP1-like partner protein of FBL17 and discuss a possible mechanism how SCFFBL17 may regulate cell division during male gametogenesis.
PMCID: PMC2651519  PMID: 19277118
22.  Spatial and temporal localization of homogalacturonans in Hyacinthus orientalis L. ovule cells before and after fertilization 
Plant Cell Reports  2014;34:97-109.
Key message
The composition of homogalacturonans (HGs) in the ovule and the female gametophyte cell walls was shown to be rearranged dynamically during sexual reproduction ofH. orientalis.
In angiosperms, homogalacturonans (HGs) play an important role in the interaction between the male gametophyte and the pistil transmitting tract, but little is known about the participation of these molecules at the final stage of the progamic phase and fertilization. The aim of our study was to perform immunocytochemical localization of highly (JIM7 MAb) and weakly (JIM5 MAb) methyl esterified and Ca2+-associated HG (2F4 MAb) in the ovule and female gametophyte cells of Hyacinthus orientalis before and after fertilization. It was found that pollination induced the rearrangement of HG in (1) the micropylar canal of the ovule, (2) the filiform apparatus of the synergids, and (3) the region of fusion between sperm cells and their target cells. Fertilization led to further changes in pectin composition of these three regions of the ovule. A new cell wall was synthesized around the zygote with a characteristic pattern of localization of all examined HG fractions, which we called “sporoderm-like”. The developing endosperm prepared for cellularization by synthesizing highly methyl-esterified HG, which was stored in the cytoplasm. Pollination- and fertilization-induced changes in the composition of the HG in the micropyle of the ovule and the apoplast of female gametophyte cells are discussed in the context of: (1) micropylar pollen tube guidance, (2) preparation of the egg cell and the central cells for fusion with sperm cells, and (3) the polyspermy block.
PMCID: PMC4282716  PMID: 25292437
Homogalacturonan (HG); Pectin; Ovule; Embryo sac; Hyacinthus orientalis
23.  The Structural Variation Is Associated with the Embryonic Lethality of a Novel Red Egg Mutant Fuyin-lre of Silkworm, Bombyx mori 
PLoS ONE  2015;10(6):e0128211.
Bombyx mori presents several types of egg color mutations, all of which have been extensively discussed in sericulture. While the red egg mutation has been previously observed, lethal red-egg mutants have not been reported. In the present work, the red egg mutant Fuyin-lre (Fuyin-lethal red egg) was discovered from the Fuyin germplasm resource of B. mori. This mutant features red-colored eggs and embryonic lethality. Genetic analysis showed that Fuyin-lre follows recessive inheritance, with the red egg gene re governing the egg color, and the embryonic lethality of Fuyin-lre may be caused by mutations of other genes closely linked to re. Digital gene expression (DGE) was employed to compare the transcription profiles of Fuyin and Fuyin-lre eggs after 24 and 48 h of incubation. A total of 48 differentially expressed genes followed the same expression patterns in both groups at both time points (FDR < 0.01 and log 2 Ratio ≥ 1). Further analyses indicated that 8 out of the 48 genes (including re) were closely linked to re. These 8 genes were highly expressed in wild-type Fuyin and the red egg mutant re but showed nearly absent expression in Fuyin-lre. Sequencing of the re gene confirmed that the re gene itself does not induce embryonic lethality, and structure analysis showed that the structural variation of the region where the 8 genes were located may be associated with the embryonic lethality of Fuyin-lre. The present work provides a good foundation for future studies on the mechanism of embryonic lethality and embryonic development in Fuyin-lre.
PMCID: PMC4452371  PMID: 26030868
24.  Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil 
PLoS Genetics  2009;5(8):e1000621.
Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction.
Author Summary
For successful reproduction in flowering plants, a single-celled pollen tube must rapidly extend through female pistil tissue, locate female gametes, and deliver sperm. Pollen tubes undergo a dramatic transformation while growing in the pistil; they grow faster compared to tubes grown in vitro and become competent to perceive and respond to navigation cues secreted by the pistil. The genes expressed by pollen tubes in response to growth in the pistil have not been characterized. We used a surgical procedure to obtain large quantities of uncontaminated pollen tubes that grew through the pistil and defined their transcriptome by microarray analysis. Importantly, we identify a set of genes that are specifically expressed in pollen tubes in response to their growth in the pistil and are not expressed during other stages of pollen or plant development. We analyzed mutants in 33 pollen tube–expressed genes using a sensitive series of pollen function assays and demonstrate that seven of these genes are critical for pollen tube growth; two specifically disrupt growth in the pistil. By identifying pollen tube genes induced by the pistil and describing a mutant analysis scheme to understand their function, we lay the foundation for functional genomic analysis of pollen–pistil interactions.
PMCID: PMC2726614  PMID: 19714218
25.  Mode of Pollen-Tube Growth in Pistils of Myrica rubra (Myricaceae): A Comparison with Related Families 
Annals of Botany  2006;97(1):71-77.
• Background and Aims It is generally known that fertilization is delayed for more than a few weeks after pollination in Fagales. Recent studies showed that, during that period, pollen tubes grew in pistils in close association with the development of the ovule in a five-step process in Casuarina (Casuarinaceae) and a four-step process in Alnus (Betulaceae). The number of pollen tubes was reduced from many to one, a fact suggesting that delayed fertilization plays a role for gametophyte selection. Myrica (Myricaceae) also shows delayed fertilization for >2 weeks after pollination, but nothing is known of how pollen tubes grow in the pistil during that period.
• Methods Pollen-tube growth and the development of the ovule in pistils was investigated by fluorescent and scanning electron microscopy and analysis of microtome sections of the pistils.
• Key Results Developmental study of the pollen-tube growth in the pistil of M. rubra showed that the tip of the pollen tube was branched or lay in a zigzag pattern in the upper space of the ovarian locule or near the tip of the integument, and subsequently was swollen on the nucellar surface. Such morphological changes indicate that the pollen-tube growth was temporarily arrested before fertilization. The pollen-tube growth in M. rubra can therefore be summarized as occurring in three steps: (1) from the stigma to the ovarian locule; (2) from the ovarian locule to the nucellar surface; and (3) from the nucellar surface to the embryo sac.
• Conclusion Myrica differs from other families in that the pollen tubes arrest their growth on the nucellar surface, probably digesting nutrient from nucellar cells. There is little information on five other families of Fagales. An extensive study is needed to better understand the diversity and function of the mode of pollen-tube growth within the order.
PMCID: PMC2803377  PMID: 16291781
Fagales; fertilization; micropyle; Myrica; Myricaceae; pollen-tube growth

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