Fertilization In Higher Plants Research Articles

Self-incompatibility requires GPI anchor remodeling by the poppy PGAP1 ortholog HLD1

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are tethered to the outer leaflet of the plasma membrane where they function as key regulators of a plethora of biological processes in eukaryotes. Self-incompatibility (SI) plays a pivotal role regulating fertilization in higher plants through recognition and rejection of "self" pollen. Here, we used Arabidopsis thaliana lines that were engineered to be self-incompatible by expression of Papaver rhoeas SI determinants for an SI suppressor screen. We identify HLD1/AtPGAP1, an ortholog of the human GPI-inositol deacylase PGAP1, as a critical component required for the SI response. Besides a delay in flowering time, no developmental defects were observed in HLD1/AtPGAP1 knockout plants, but SI was completely abolished. We demonstrate that HLD1/AtPGAP1 functions as a GPI-inositol deacylase and that this GPI-remodeling activity is essential for SI. Using GFP-SKU5 as a representative GPI-AP, we show that the HLD1/AtPGAP1 mutation does not affect GPI-AP production and targeting but affects their cleavage and release from membranes invivo. Our data not only implicate GPI-APs in SI, providing new directions to investigate SI mechanisms, but also identify a key functional role for GPI-AP remodeling by inositol deacylation in planta.

Isolation of Egg and Sperm Cells from Bauhinia blakeana

Isolated sperm cells and egg cells provide useful materials for the study of fertilization in higher plants, under controlled conditions, in the absence of somatic tissue. Viable egg cells from Bauhinia blakeana were isolated using enzymatic digestion and mechanical dissection. Ovules were digested with enzymes for 40–50 min and then transferred to a solution without enzymes for dissection. The three cells of the egg apparatus (i.e., the egg cell and two synergids) were released from a cut ovule by pushing the micropylar region of the ovule with a dissecting needle. Typically, 7–10 egg cells were isolated within 1 h. B. blakeana pollen is bicellular and contains only a vegetative and generative cell at anthesis. Sperm cells were isolated using an in vivo–in vitro method. The isolation of B. blakeana egg and sperm cells potentially provides a basis for in vitro fertilization of this woody dicotyledonous plant and materials for molecular biological investigations of fertilization mechanisms.

Advances in the study of egg activation of higher plants.

Fertilization in higher plants induces many structural and physiological changes in the fertilized egg, and represents the transition from the haploid female gamete to the diploid zygote, the first cell of a sporophyte. Some changes are induced extremely rapidly following fusion with sperm cells and are the preclusions of egg activation. This review focuses on the early changes that occur in the egg after fusion with sperm cells, but before nuclear fusion. Reported changes include cell shrinkage, cell wall formation, polarity change, oscillation in Ca2+ concentration, and DNA synthesis. In addition, the current understanding of egg activation is summarized and the possible functional relevance of the changes is explored.

Global proteomic and phosphoproteomic analysis of the premature maize anther

Reversible phosphorylation plays a crucial role in regulating protein activities and functions. Sexual reproduction directly affects yield of most agricultural crops. As the male reproductive organ, anther generates microspores (pollen), delivering gametes (sperms) to complete double fertilization in higher plants. Here, we took the advantage of Nano UHPLC-MS/MS to analyze maize (Zea mays, B73) early anthers at proteomic and phosphoproteomic levels, to explore the protein and phosphorylation modification regulatory networks controlling maize anther development. Our proteomic analysis identified 3 016 unique peptides, belonging to 1 032 maize proteins. MapMan analysis revealed variously potential proteins associated with maize anther development, such as receptor-like kinases (GRMZM2G082823_P01 and GRMZM5G805485_P01). Using phospho-peptides enriched by TiO2 affinity chromatography, our phosphoproteomic analysis detected 257 phospho-peptides from 210 phosphoproteins, discovering 223 phosphosites. Compared to the 86 maize phosphoproteins collected in the Plant Protein Phosphorylation Data Base (P3DB), we found that 203 phosphoproteins and 218 phosphosites were not revealed before. Further bioinformatics analysis revealed that phosphorylation of 14-3-3 proteins, kinases, phosphatases, transcription factors, cell cycle and chromatin structure related proteins might play important roles in regulating normal anther development in maize. Our findings not only enlarged the maize phosphoproteome data, but also provided information for analyzing the molecular mechanism controlling maize anther development at genetic and biochemical levels.

Late progamic phase and fertilization affect calreticulin expression in the Hyacinthus orientalis female gametophyte.

Key messageCalreticulin expression is upregulated during sexual reproduction ofHyacinthus orientalis, and the protein is localized both in the cytoplasm and a highly specialized cell wall within the female gametophyte.Several evidences indicate calreticulin (CRT) as an important calcium (Ca2+)-binding protein that is involved in the generative reproduction of higher plants, including both pre-fertilization and post-fertilization events. Because CRT is able to bind and sequester exchangeable Ca2+, it can serve as a mobile intracellular store of easily releasable Ca2+ and control its local cytosolic concentrations in the embryo sac. This phenomenon seems to be essential during the late progamic phase, gamete fusion, and early embryogenesis. In this report, we demonstrate the differential expression of CRT within Hyacinthus female gametophyte cells before and during anthesis, during the late progamic phase when the pollen tube enters the embryo sac, and at the moment of fertilization and zygote/early endosperm activation. CRT mRNA and the protein localize mainly to the endoplasmic reticulum (ER) and Golgi compartments of the cells, which are involved in sexual reproduction events, such as those in sister synergids, the egg cell, the central cell, zygote and the developing endosperm. Additionally, immunogold research demonstrates selective CRT distribution in the filiform apparatus (FA), a highly specific component of the synergid cell wall. In the light of our previous data showing the total transcriptional activity of the Hyacinthus female gametophyte and the results presented here, we discuss the possible functions of CRT with respect to the critical role of Ca2+ homeostasis during key events of sexual plant reproduction. Moreover, we propose that the elevated expression of CRT within the female gametophyte is a universal phenomenon in the cells involved in double fertilization in higher plants.

Molecular Mechanisms behind the Male-Female Interaction in Fertilization in Higher Plants

In angiosperm, fertilization starts from pollen adhesion, hydration, and germination. Pollen tubes penetrate the stigma, grow into the transmitting tract, and then onto the funiculi by breaking through septums. Finally, one pollen tube is guided to enter micropyle and release two sperm cells that fuse with the egg cell and the central cell respectively to complete fertilization. Notably, the sperm cells of angiosperm, lack of flagellum, are transported into the embryo sac with the help of pollen tubes. It is well received that fertilization is tightly controlled by the male-female interaction at different stages. For example, the pollen tube recognizes and responds to the signals from the ovule so that it can grow into the tiny micropylar opening, and know the proper time to release sperm cells. Meanwhile, sperm cells fuse with the egg and the central cell respectively after signals from both sides are perceived. Therefore, the fertilization depends on the successful recognition and interaction between male and female gametophytes at different developmental stages. This review summarizes recent progress on these interactions during fertilization.

The language of GABA in pollen tube growth and guidance

The achievement of double fertilization in higher plants requires the successful transport of sperm cells to the female gametes, the ovules. Pollen tubes, the tubular structure protruding from pollens, carrying the sperms play an important role in this process. How a pollen tube precisely guides its direction to gain its goal is of mystery. Previous investigation indicated that multiple signal clues from the pistils function as the route signs to regulate the pathway of pollen tube growth. Among the signal clues, γ-aminobutyric acid (GABA) seems to be universal. Its gradient concentration has been found both in tobacco and Arabidopsis pollen tube guidance. In the communication of pollens and pistils, what on earth GABA tells pollen tubes is of great interest. The GABA receptors on the pollen membrane are thought to be the hinge in the language conversation. In this review, the mechanism of GABA gradient formation is investigated. The possible GABA receptor on the pollen membrane is examined and its function is discussed. To decipher the possible language of GABA in pollen tube growth and guidance, multiple methods are needed. The combination of transcriptome and proteomics assay is expected to unveil the secret.

Reproductive Dialog

Fertilization in higher plants requires intricate signaling between the male and female. The molecular details underlying this communication are of great interest, not only to understand plant reproduction but also to guide efforts in crossing plant species to generate new hybrids. On page 656 of this issue, Escobar-Restrepo et al. (1) show that cells in the female ovule express a protein—a type of receptor that is widely known to mediate cell-cell communication—that is required for fertilization. The unknown ligand(s) for the receptor may be present on or secreted by the pollen tube. Such a receptor-ligand interaction could control species-specific reproduction.

In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.

An Ankyrin Repeat-Containing Protein, Characterized as a Ubiquitin Ligase, Is Closely Associated with Membrane-Enclosed Organelles and Required for Pollen Germination and Pollen Tube Growth in Lily

Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth.

Anther-specific genes, which expressed through microsporogenesis, are temporally and spatially regulated in model legume, Lotus japonicus

Pollen germination and pollen tube elongation are important for pollination and fertilization in higher plants. To date, several pollen-specific genes have been isolated and characterized. However, there is little information about the precise spatial and temporal expression pattern of pollen-specific genes in higher plants. In our previous study, we identified 132 anther-specific genes in the model legume Lotus japonicus by using cDNA microarray analysis, though their precise expression sites in the anther tissues were not determined. In this study, by using in situ hybridization experiments, we determined the spatial and temporal expression sites of 46 anther-specific genes (ca. 35%), which were derived from two groups, cluster I-a and cluster II-a, according to flower developmental stages. In the case of the genes grouped into cluster I-a, thirteen clones were characterized. The specific hybridized signals were varied among the clones, and were observed in tapetum cells, microspores, and anther walls at the early developmental stage of anther tissues. In the case of the genes classified into cluster II-a, we used thirty three different cDNA clones encoding primary and secondary metabolism-related proteins, cell wall reconstruction-related proteins, actin reorganization-related proteins, and sugar transport-related proteins, etc., as a probe. Interestingly, all genes in these thirty three clones examined were specifically expressed in the bicellular pollen grains, though the signal intensity was varied among clones. From the data of the cluster II-a genes, the mRNAs related to pollen germination and pollen tube elongation were specifically transcribed and preserved in mature pollen grains.

A calcium influx is triggered and propagates in the zygote as a wavefront during in vitro fertilization of flowering plants.

In this paper, we report direct measurement of an influx of extracellular Ca(2+) induced by gamete fusion in flowering plants. This result was obtained during maize in vitro fertilization with the use of an extracellular Ca(2+)-selective vibrating probe. Ca(2+) influx recorded at the surface of isolated egg cells, with or without adhesion of a male sperm cell, was close to zero and stable over time. Gamete fusion, however, triggered a Ca(2+) influx in the vicinity of the sperm entry site with a delay of 1.8 +/- 0.6 sec. The Ca(2+) influx spread subsequently through the whole egg cell plasma membrane as a wavefront, progressing at an estimated rate of 1.13 micrometer.(-1). Once established, Ca(2+) influx intensities were sustained, monotonic and homogeneous over the whole egg cell, with an average peak influx of 14.92 pmol .cm(-2).(-1) and an average duration of 24.4 min. The wavefront spread of channel activation correlates well with the cytological modifications induced by fertilization, such as egg cell contraction, and with the cytosolic Ca(2+) ((c)[Ca(2+)]) elevation previously reported. Calcium influx was inhibited effectively by gadolinium, possibly implicating mechanosensitive channels. Furthermore, artificial influxes created by incubation with Ca(2+) ionophores mimicked some aspects of egg activation. Taken together, these results suggest that, during fertilization in higher plants, gamete membrane fusion starts the first embryonic events by channel opening and Ca(2+) influx. In turn, (c)[Ca(2+)] may work as a trigger and possibly a space and time coordinator of many aspects of egg activation.

The components of the sexual reproduction process and the pathway toin Vivofertilization in higher plants

Summary Successful higher plant reproduction depends upon the development of functional reproductive structures and viable gametes in the flowers, transfer of viable pollen onto a receptive and compatible stigma, pollen germination, directed pollen tube growth to the ovule, gamete delivery and nuclear fusion. In higher plants, two nuclear fusions must take place — one sperm nucleus fusing with the egg nucleus to form the zygote, the other sperm fusing with the polar nuclei to form the endosperm. Seed development to maturity may follow.

Critical Discussion on Abortive Interspecific Crosses in Brassica

AbstractIn most interspecific hybridization among diploid Brassica species (n = 8, 9, 10), few or no hybrids are obtained, and the three crosses of tetraploid (n = 17, 18, 19) by diploid species are usually only partially successful, especially in one of the reciprocal crosses. Such a cross‐incompatibility complex is explained in this paper by a polar‐nuclei activation (PNA) hypothesis which proposes two reversible functions, a normal reproduction and a sexual barrier, of double fertilization in higher plants. The first, i.e. the relative activating value (AV) of diploid species, was estimated as 1.0–3.5, whereas that of tetraploid species was 2.7–5.2. In addition, crosses with a polar nuclei activation index (AI) of about 15–87 % were compatible but those beyond the two limits were incompatible, 50 % of those showing normal seed development after self‐pollination.

Molecular basis of cell recognition during fertilization in higher plants.

The molecular basis of recognition between plant cells is incompletely understood. Some principles established for recognition between animal cells may well apply to plant cell recognition, although, in contrast to animal cells, plant cells are encased by cell walls that play an active role in plant cell-cell recognition. The interaction that controls fertilization in flowering plants involves recognition between pollen or pollen tubes and the female sexual tissues. In many flowering plant families, self-incompatibility (S) genes operate to prevent inbreeding. In plants that have gametophytically controlled self-incompatibility, recognition of common S alleles in pollen tube and style results in arrest of pollen tube growth within the style. Self-incompatibility therefore provides a model cell-cell recognition system that is genetically defined. We have taken two approaches to defining cell recognition involved in gametophytic self-incompatibility in Nicotianas alata. Firstly, we have established the major features of the pollen tube wall and the matrix of the style transmitting tissue that are in contact with the growing pollen tube. Secondly, we have established the nature of style glycoproteins that are associated with the S genotype and have initiated a program to clone the genes coding for the protein component of these glycoproteins. Analyses of the pollen tube are consistent with the major polymers being a (1----3)-beta-D-glucan (callose) and a (1----5)-alpha-L-arabinan. The pollen tube has two distinct layers: gold immunocytochemistry using a monoclonal antibody directed to terminal alpha-L-arabinosyl residues shows the binding is confined to the outer layers. The major component of the extracellular matrix of the style transmitting tissue is a family of proteoglycans, the arabinogalactan-proteins. A major glycoprotein that segregates with the S2 allele is present in extracts of mature styles. This component has a high pI (greater than 9.5) and an apparent molecular weight of 32 X 10(3). It is not present in extracts of immature styles of N. alata genotypes bearing the S2 allele, or in extracts from other organs of N. alata or styles of other members of the Solanaceae. The isolated glycoprotein is an effective inhibitor of in vitro pollen tube growth. This evidence suggests that the S2-associated glycoprotein is either the product of the S2 allele, or a gene closely associated with the S gene. We have prepared a cDNA library from styles of one genotype and are screening this library with mRNA from mature and immature styles.(ABSTRACT TRUNCATED AT 400 WORDS)