Sexual Reproduction In Higher Plants Research Articles

The Role of Reactive Oxygen Species in the In Vitro Germination and Growth of the Petunia (Petunia hybrida E. Vilm.) Male Gametophyte

The in vitro growth of the pollen tube (PT), an object of comprehensive and intensive research, is a model for studying the mechanisms of sexual reproduction in higher plants. We have studied the potential role of reactive oxygen species (ROS) in the in vitro germination and growth maintenance of the petunia (Petunia hybrida E. Vilm.) male gametophyte. The exogenous treatment with H2O2 influences the PT germination and polar growth in vitro. The addition of H2O2 to culture medium increases both the percentage of pollen grain germination and the PT length in the case of long cultivation, but inhibits both processes during the first hour of cultivation. This suggests that endogenous ROS play a decisive role in the early stages of pollen germination, with the sensitivity to endogenous ROS emerging later over the course of their growth. The addition of diphenylene iodonium chloride (DPI), a NADPH oxidase inhibitor, considerably decreases both the germination and the growth of the petunia male gametophyte at low concentrations (0.1 μM), and completely arrests the growth at high concentrations (1 μM). ROS are necessary for polar growth of the petunia male gametophyte; they are secreted in the early stages of pollen grain activation and are further localized to the initiation of the PT, mainly in the PT apical part, during polar growth, as confirmed with the help of intravital fluorescence microscopy.

First report of <i>Alternaria alternata</i> causing flower blight on <i>Camellia sinensis</i> in Hefei, China

Flowers are an essential organ for sexual reproduction of higher plants. Severe lesions along with flower blight on tea (<italic>Camellia sinensis</italic>) plants were observed in the experimental tea plantation located in Hefei (China). The pathogens isolated from diseased flowers matched the morphological peculiarity of <italic>Alternaria alternata</italic>. The species characteristics of <italic>A. alternata</italic> were further confirmed by both pathogenicity tests and multi-gene phylogenetic analyses by using internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB). The combined phylogeny analysis using sequences derived from the ITS, GAPDH and TUB showed that the isolated pathogens belong to the genus <italic>Alternaria</italic>. Pathogenicity tests conducted on healthy tea flowers and leaves manifested typical symptoms of flower blight while weaker symptoms of leaf spot, demonstrating the<italic> A. alternata</italic> isolates were the causal agents of flower blight disease on tea plants. This fungus is first reported as a pathogen causing flower blight on <italic>C. sinensis</italic> in this study.

Secretory Vesicles Targeted to Plasma Membrane During Pollen Germination and Tube Growth.

Pollen germination and pollen tube growth are important biological events in the sexual reproduction of higher plants, during which a large number of vesicle trafficking and membrane fusion events occur. When secretory vesicles are transported via the F-actin network in proximity to the apex of the pollen tube, the secretory vesicles are tethered and fused to the plasma membrane by tethering factors and SNARE proteins, respectively. The coupling and uncoupling between the vesicle membrane and plasma membrane are also regulated by dynamic cytoskeleton, proteins, and signaling molecules, including small G proteins, calcium, and PIP2. In this review, we focus on the current knowledge regarding secretory vesicle delivery, tethering, and fusion during pollen germination and tube growth and summarize the progress in research on how regulators and signaling molecules participate in the above processes.

DNA methylation dynamics of sperm cell lineage development in tomato.

During the sexual reproduction of higher plants, DNA methylation and transcription are broadly changed to reshape a microspore into two sperm cells (SCs) and a vegetative cell (VC). However, when and how the DNA methylation of SCs is established remains not fully understood. Here we investigate the DNA methylation (5mC) dynamics of SC lineage and the VC in tomato using whole-genome bisulfite sequencing. We find the asymmetric division of the microspore gives its two daughter cells differential methylome. Compared with the generative cell (GC), the VC is hypomethylated at CG sites while hypermethylated at CHG and CHH sites, with the majority of differentially methylation regions targeted to transposable elements (TEs). SCs have a nearly identical DNA methylome to the GC, suggesting that the methylation landscape in SCs may be pre-established following the asymmetric division or inherited from the GC. The random forest classifier for predicting gene and TE expression shows that methylation within the gene body is a more powerful predictor for gene expression. Among all tested samples, gene and TE expression in the microspore may be more predictable by DNA methylation. Our results depict an intact DNA methylome landscape of SC lineage in higher plants, and reveal that the impact of DNA methylation on transcription is variant in different cell types.

Maize male sterile 33 encodes a putative glycerol-3-phosphate acyltransferase that mediates anther cuticle formation and microspore development

BackgroundThe anther cuticle, which is primarily composed of lipid polymers, is crucial for pollen development and plays important roles in sexual reproduction in higher plants. However, the mechanism underlying the biosynthesis of lipid polymers in maize (Zea mays. L.) remains unclear.ResultsHere, we report that the maize male-sterile mutant shrinking anther 1 (sa1), which is allelic to the classic mutant male sterile 33 (ms33), displays defective anther cuticle development and premature microspore degradation. We isolated MS33 via map-based cloning. MS33 encodes a putative glycerol-3-phosphate acyltransferase and is preferentially expressed in tapetal cells during anther development. Gas chromatography-mass spectrometry revealed a substantial reduction in wax and cutin in ms33 anthers compared to wild type. Accordingly, RNA-sequencing analysis showed that many genes involved in wax and cutin biosynthesis are differentially expressed in ms33 compared to wild type.ConclusionsOur findings suggest that MS33 may contribute to anther cuticle and microspore development by affecting lipid polyester biosynthesis in maize.

Arabidopsis thaliana NOP10 is required for gametophyte formation.

The female gametophyte is crucial for sexual reproduction of higher plants, yet little is known about the molecular mechanisms underlying its development. Here, we report that Arabidopsis thaliana NOP10 (AtNOP10) is required for female gametophyte formation. AtNOP10 was expressed predominantly in the seedling and reproductive tissues, including anthers, pollen grains, and ovules. Mutations in AtNOP10 interrupted mitosis of the functional megaspore during early development and prevented polar nuclear fusion in the embryo sacs. AtNOP10 shares a high level of amino acid sequence similarity with Saccharomyces cerevisiae (yeast) NOP10 (ScNOP10), an important component of the H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) implicated in 18S rRNA synthesis and rRNA pseudouridylation. Heterologous expression of ScNOP10 complemented the mutant phenotype of Atnop10. Thus, AtNOP10 influences functional megaspore mitosis and polar nuclear fusion during gametophyte formation in Arabidopsis.

Hydrogel control of water uptake by pectins during in vitro pollen hydration of Eucalyptus globulus.

Upon pollination, dehydrated pollen grains take water out of the stigma surface, an event that constitutes the first functional checkpoint of sexual reproduction in higher plants. Little is known about possible functional connections between rehydration speed and further steps of fertilization. Here we addressed the mechanisms of water uptake control by dehydrated pollen grains. Because dehydrated cells have no energy-driven active mechanism such as membrane-based osmoregulation for controlling water movement, we tested the hypothesis that another mechanism might exist, namely, the use of hydrogel-behaving molecules. We developed an imaging protocol to visualize and quantify the rate of water entry into pollen grains of Eucalyptus globulus and tested the influence of different treatments linked to hydrogel-behaving molecules. We complemented these analyses by immunostaining pectins in the pollen grain with monoclonal antibodies JIM5 and JIM7. Water entry seemed to proceed exclusively through the germination apertures of the pollen grain, and the changes observed in different hydration media are compatible with hydrogel behavior. When JIM5 and JIM7 were used to characterize pectins on the germination apertures during hydration, pectin localization and esterification changed during hydration and were affected by the hydration solutions. These results suggest that chemical modification of the pectins may change their hydrogel behavior, thus modifying the hydration speed. The hydrogel behavior of pectins and pectin localization on apertures strongly suggest that pectins act like "valves" for water entry, enabling a regulated process of water uptake into the dehydrated pollen grain. We propose that this regulation evolved in terms of achieving the correct self-organization of molecules and cellular components to resume metabolism and pollen tube growth, especially in species that are subject to demanding environmental water stress.

Role of auxin and cytokinin in the regulation of the actin cytoskeleton in the in vitro germinating male gametophyte of petunia

Pollen tubes germinating in vitro, which has become in recent years the object of detail and intensive research, is a model for studying the mechanisms of sexual reproduction of higher plants. The potential role of the actin cytoskeleton (F-actin) in the transduction of hormonal signals in the progamic phase of fertilization was studied on in vitro germinating male gametophyte of petunia (Petunia hybrida L.). It was established for the first time that treatments with phytohormones, such as auxin (IAA) and cytokinin (kinetin), affected germination and polar growth of the pollen tubes, e.g., stimulated or inhibited these processes, and also induced the actin cytoskeleton rearrangement. The addition of IAA to nutrient medium increased the content of F-actin in the apical and subapical zones of pollen tubes and, as it is supposed, is responsible for the stimulation of their polar growth due to the intensification of the cytoplasm flow and apically directed vesicle transport. The inhibitor of actin polymerization, latrunculin B, disturbed actin cytoskeleton spatial organization, decreased the content of endogenous IAA, and, as a consequence, retarded germination and/or blocked male gametophyte polar growth. In contrast, kinetin suppressed pollen tube growth, reducing the content of polymer actin along the entire tube length. In pollen tubes germinating in the presence of latrunculin B for 4 h, the content of cytokinins was not significantly changed as compared with control. These results allow a conclusion that auxin and cytokinin are involved in the regulation of pollen tube polar growth via their effects on actin polymerization and spatial organization.

Pollen Lipidomics: Lipid Profiling Exposes a Notable Diversity in 22 Allergenic Pollen and Potential Biomarkers of the Allergic Immune Response

Background/AimPollen grains are the male gametophytes that deliver sperm cells to female gametophytes during sexual reproduction of higher plants. Pollen is a major source of aeroallergens and environmental antigens. The pollen coat harbors a plethora of lipids that are required for pollen hydration, germination, and penetration of the stigma by pollen tubes. In addition to proteins, pollen displays a wide array of lipids that interact with the human immune system. Prior searches for pollen allergens have focused on the identification of intracellular allergenic proteins, but have largely overlooked much of the extracellular pollen matrix, a region where the majority of lipid molecules reside. Lipid antigens have attracted attention for their potent immunoregulatory effects. By being in close proximity to allergenic proteins on the pollen surface when they interact with host cells, lipids could modify the antigenic properties of proteins.Methodology/Principal FindingsWe performed a comparative pollen lipid profiling of 22 commonly allergenic plant species by the use of gas chromatography-mass spectroscopy, followed by detailed data mining and statistical analysis. Three experiments compared pollen lipid profiles. We built a database library of the pollen lipids by matching acquired pollen-lipid mass spectra and retention times with the NIST/EPA/NIH mass-spectral library. We detected, identified, and relatively quantified more than 106 lipid molecular species including fatty acids, n-alkanes, fatty alcohols, and sterols. Pollen-derived lipids stimulation up-regulate cytokines expression of dendritic and natural killer T cells co-culture.Conclusions/SignificanceHere we report on a lipidomic analysis of pollen lipids that can serve as a database for identifying potential lipid antigens and/or novel candidate molecules involved in allergy. The database provides a resource that facilitates studies on the role of lipids in the immunopathogenesis of allergy. Pollen lipids vary greatly among allergenic species and contain many molecules that have stimulatory or regulatory effects on immune responses.

Techniques of cell type-specific transcriptome analysis and applications in researches of sexual plant reproduction

In higher plants, specific cell differentiation and fate decision are controlled by differential gene expression. Cell type-specific transcriptome analysis has become an important tool for investigating cell regulatory mechanisms. In recent years, many different techniques have been developed for the isolation of specific cells and the subsequent transcriptome analysis, and considerable data are available regarding the transcriptional profiles of some specific cells. These cell type-specific transcriptome analyses hold significant promise for elucidating the gene expression linked to cellular identities and functions, and are extraordinarily important for research in functional genomics and systems biology aimed toward basic understanding of molecular networks and pathway interactions. Moreover, to reveal the critical mechanisms about sexual plant reproduction, the gamete and embryo cells have long been treated as good subjects for cell-specific transcriptome analysis, and there has been important progress in recent decades. In this review, we summarize current technologies in cell type-specific transcriptome analysis and review the applications of these technologies in research into the mechanisms of sexual reproduction in higher plants.

Isolation of Gametophytic Cells and Identification of Their Cell-Specific Markers in Torenia fournieri, T. concolor and Lindernia micrantha

Gametophytic cells in the female embryo sac, the egg, the central and the synergid cells have unique roles in sexual reproduction in higher plants. To investigate their cell-specific features, an efficient method of distinguishing and isolating each gametophytic cell is required. By testing the activities of several cellulase enzymes, we modified our previous cell isolation method and succeeded in isolating each cell from the Torenia and Lindernia species. We also established genetic markers that show specific expression patterns in each gametophytic cell. These markers, together with the cell isolation method, will be useful for analyzing the function of gametophytic cells.

A compartmental model analysis of integrative and self-regulatory ion dynamics in pollen tube growth.

Sexual reproduction in higher plants relies upon the polarised growth of pollen tubes. The growth-site at the pollen tube tip responds to signalling processes to successfully steer the tube to an ovule. Essential features of pollen tube growth are polarisation of ion fluxes, intracellular ion gradients, and oscillating dynamics. However, little is known about how these features are generated and how they are causally related. We propose that ion dynamics in biological systems should be studied in an integrative and self-regulatory way. Here we have developed a two-compartment model by integrating major ion transporters at both the tip and shank of pollen tubes. We demonstrate that the physiological features of polarised growth in the pollen tube can be explained by the localised distribution of transporters at the tip and shank. Model analysis reveals that the tip and shank compartments integrate into a self-regulatory dynamic system, however the oscillatory dynamics at the tip do not play an important role in maintaining ion gradients. Furthermore, an electric current travelling along the pollen tube contributes to the regulation of ion dynamics. Two candidate mechanisms for growth-induced oscillations are proposed: the transition of tip membrane into shank membrane, and growth-induced changes in kinetic parameters of ion transporters. The methodology and principles developed here are applicable to the study of ion dynamics and their interactions with other functional modules in any plant cellular system.

Comparative Detection of Calcium Fluctuations in Single Female Sex Cells of Tobacco to Distinguish Calcium Signals Triggered by in vitro Fertilization

Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca(2+)-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca(2+) in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca(2+) signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 microM Fluo-3 for 30 min at 30 degrees C. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca(2+)](cyt) of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.

Towards a Comprehensive Understanding of Molecular Mechanisms of Sexual Reproduction in Higher Plants

Sexual reproduction in higher plants includes a key phase for producing male and female gametes (i.e. microsporogenesis, microgametogenesis, megasporogenesis and megagametogenesis), in addition to the ensuing pollination and fertilization. Development of the gametes is regulated by gametophytic and sporophytic gene expression ( McCormick 2004 ); for example, gametophytic microspores are infl uenced by gene expression in sporophytic tapetum cells of anthers. Such networks of gene expression during gamete development are quite important for the understanding of sexual reproduction, because failure of gene regulation often results in a defect of fertilization, which is known as male and/or female sterility. To date, many reproductive tissuespecifi c genes have been identifi ed and characterized ( Taylor and Hepler 1997 , Endo et al. 2004 , Kagi and Gross-Hardt 2007 ). However, the total picture of gene regulation of events during sexual reproduction is still not clear. This special issue consists of four original articles about transcriptome analysis focused on male gamete development, two original articles about mutant analysis of male or female gamete development, and one review article about male sterility. All the papers take up a common theme about ‘gene regulation in plant reproduction’, and should help towards a comprehensive understanding of sexual reproduction in higher plants. Tapetum cells, which are the innermost cell layer of the anther wall, are important for feeding nutrients to microspores, and pollen maturation is accompanied by the characteristic gene expression of microspore/pollen and tapetum. Therefore, analyses of separated transcriptomes of microspore/pollen and tapetum are necessary to gain an understanding of male gametophyte development. Laser microdissection (LM) is a powerful tool for isolating specifi c cells from complicated plant tissues ( Nakazono et al. 2003 ), and can be applied to these separated transcriptomes. In this issue, authors of four papers have referred to the LM 44K-microarray (44K-LMM) of microspore/pollen and tapetum in japonica rice. First, Suwabe and colleagues established the 44K-LMM with amplifi ed labeled RNAs isolated from microspore/pollen and tapetum cells, which were physically separated from a cross-section of rice anthers. In order to validate the 44K-LMM data, they compared the 44K-LMM data with that of 156 previously characterized anther-specifi c genes, which were identifi ed by Endo et al. (2004) . From this validation, it was demonstrated that the 44K-LMM is highly reliable, and can be used for further comprehensive analysis of gene networks in the development of the male gametophyte (pp. 1407–1416). Secondly, Hobo and colleagues characterized 44K-LMM data by using in silico analyses (cluster analysis, gene ontology, and searching gene regulatory cis -elements and mutant phenotypes). Interestingly, synchronous gene expression between the microspore and tapetum was observed as a novel characteristic in these transcriptomes. Further detailed analyses will reveal the signifi cance of this synchronous expression in the development of the male gamete. In addition, about half of the Tos17 insertion lines in microspore/pollenor tapetumspecifi c genes showed sterile or low fertility phenotypes, indicating that these genes were necessary for the development of anther tissues (pp. 1417–1428). These two fundamental papers clearly indicate that these precise transcriptome data sets of male gametophyte development are useful for understanding various phenomena in the anther, and will shed light on new research fi elds of plant reproduction. The relationship between phytohormones and microspore/ pollen and tapetum transcriptomes is one of the most interesting fi ndings using this 44K-LMM. The phytohormones are important for plant growth, development, differentiation, etc. However, only a few functions in microspore/pollen and tapetum development are known. To date, many genes encoding the regulation of biosynthesis, perception and signal transduction of several phytohormones have been identifi ed and characterized ( Guo and Ecker 2004 , Guilfoyle and

Sexual Reproduction in Higher Plants I: Fertilization and the Initiation of Zygotic Program

Sexual plant reproduction is a critical developmental step in the life cycle of higher plants, to allow maternal and paternal genes to be transmitted in a highly regulated manner to the next generation. During evolution, a whole set of signal transduction machinery is developed by plants to ensure an error-free recognition between male and female gametes and initiation of zygotic program. In the past few years, the molecular machineries underlying this biological process have been elucidated, particularly on the importance of synergid cells in pollen tube guidance, the Ca(++) spike as the immediate response of fertilization and the epigenetic regulation of parental gene expressions in early zygotic embryogenesis. This review outlines the most recent development in this area.

Self-incompatibility inPapaver: signalling to trigger PCD in incompatible pollen

Sexual reproduction in higher plants uses pollination, involving interactions between pollen and pistil. Self-incompatibility (SI) prevents self-fertilization, providing an important mechanism to promote outbreeding. SI is controlled by the S-locus; discrimination occurs between incompatible pollen, which is rejected, while compatible pollen can achieve fertilization. In Papaver rhoeas, S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to effect rapid inhibition of tip growth. This self-incompatible interaction triggers a Ca(2+)-dependent signalling cascade. SI-specific events triggered in incompatible pollen include rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases, and activation of a MAPK. It has recently been shown that programmed cell death (PCD) is triggered by SI. This provides a precise mechanism for the specific destruction of 'self' pollen. Recent data providing evidence for SI-induced caspase-3-like protease activity, and the involvement of actin depolymerization and MAPK activation in SI-mediated PCD will be discussed. These studies not only significantly advance our understanding of the mechanisms involved in SI, but also contribute to our understanding of functional links between signalling components and initiation of PCD in a plant cell. Recent data demonstrating SI-mediated modification of soluble inorganic pyrophosphatases are also described.

The function of actin-binding proteins in pollen tube growth

Pollen tube growth is a key step in sexual reproduction of higher plants. The pollen tube is a typical example of tip-growing cells and shows a polarized cytoplasm. To develop and maintain polarized growth, pollen tubes need a carefully regulated actin cytoskeleton. It is well known that actin-binding proteins are responsible for the direct control of dynamic actin filaments and serve as a link between signal transduction pathways and dynamic actin changes in determining cellular architecture. Several of these classes have been identified in pollen tubes and their detailed characterisation is progressing rapidly. Here, we aim to survey what is known about the major actin-binding proteins that affect actin assembly and dynamics, and their higher-order organisation in pollen tube growth.

Isolation of 151 Mutants that Have Developmental Defects from T-DNA Tagging

In order to understand the mechanisms underlying plant development, a necessary first step involves the elucidation of the functions of the genes, via the analysis of mutants that exhibit developmental defects. In this study, an activation tagging mutant library harboring 80,650 independent Arabidopsis transformants was generated in order to screen for developmental mutants. A total of 129 mutants manifesting dominant developmental abnormalities were isolated, and their T-DNA insertion loci were mapped. The activation of one or more genes adjacent to a T-DNA insertion locus was confirmed in eight dominant mutants. A gene adjacent to the right border was usually activated by the 35S enhancers. Interestingly, the transcriptional activation of multiple genes within a broad range was observed in one of the mutants, which raises the possibility that activation by the 35S enhancers was not limited strictly to a single gene. In order to gain a better understanding of sexual reproduction in higher plants, we isolated 22 mutants exhibiting defects in female gametophyte development, and determined their T-DNA insertion loci. We propose that this mutant population may prove useful in the further determination of the functions of genes that play important roles in plant development.

A reference map of the Arabidopsis thaliana mature pollen proteome

The male gametophyte (or pollen) plays an obligatory role during sexual reproduction of higher plants. The extremely reduced complexity of this organ renders pollen a valuable experimental system for studying fundamental aspects of plant biology such as cell fate determination, cell–cell interactions, cell polarity, and tip-growth. Here, we present the first reference map of the mature pollen proteome of the dicotyledonous model plant species, Arabidopsis thaliana. Based on two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight, and electrospray quadrupole time-of-flight mass spectrometry, we reproducibly identified 121 different proteins in 145 individual spots. The presence, subcellular localization, and functional classification of the identified proteins are discussed in relation to the pollen transcriptome and the full protein complement encoded by the nuclear Arabidopsis genome.

Aquaporins of the PIP2 class are required for efficient anther dehiscence in tobacco.

Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues. Before flower anthesis, anther and pollen dehydration takes place before the release of mature pollen at dehiscence. Aquaporins represent a class of proteins that mediates the movement of water over cellular membranes. Aquaporins of the plasmamembrane PIP2 family are expressed in tobacco (Nicotiana tabacum) anthers and may therefore be involved in the movement of water in this organ. To gain more insight into the role these proteins may play in this process, we have analyzed their localization using immunolocalizations and generated plants displaying RNA interference of PIP2 aquaporins. Our results indicate that PIP2 protein expression is modulated during anther development. Furthermore, in tobacco PIP2 RNA interference plants, anther dehydration was slower, and dehiscence occurred later when compared with control plants. Together, our results suggest that aquaporins of the PIP2 class are required for efficient anther dehydration prior to dehiscence.