Male-female communication triggers calcium signatures during fertilization in Arabidopsis

Cell-cell communication and interaction is critical during fertilization and triggers free cytosolic calcium ([Ca2+](cyto)) as a key signal for egg activation and a polyspermy block in animal oocytes. Fertilization in flowering plants is more complex, involving interaction of a pollen tube with egg adjoining synergid cells, culminating in release of two sperm cells and their fusion with the egg and central cell, respectively. Here, we report the occurrence and role of [Ca2+](cyto) signals during the entire double fertilization process in Arabidopsis. [Ca2+](cyto) oscillations are initiated in synergid cells after physical contact with the pollen tube apex. In egg and central cells, a short [Ca2+](cyto) transient is associated with pollen tube burst and sperm cell arrival. A second extended [Ca2+](cyto) transient solely in the egg cell is correlated with successful fertilization. Thus, each female cell type involved in double fertilization displays a characteristic [Ca2+](cyto) signature differing by timing and behaviour from [Ca2+](cyto) waves reported in mammals

Differential expression patterns of Arabinogalactan Proteins in Arabidopsis thaliana reproductive tissues

36 p.-6 fig.Arabinogalactan proteins (AGPs) are heavily glycosylated proteins existing in all members of the plant kingdom and are differentially distributed through distinctive developmental stages. Here, we showed the individual distributions of specific Arabidopsis AGPs: AGP1, AGP9, AGP12, AGP15, and AGP23, throughout reproductive tissues and indicated their possible roles in several reproductive processes. AGP genes specifically expressed in female tissues were identified using available microarray data. This selection was confirmed by promoter analysis using multiple green fluorescent protein fusions to a nuclear localization signal, β-glucuronidase fusions, and in situ hybridization as approaches to confirm the expression patterns of the AGPs. Promoter analysis allowed the detection of a specific and differential presence of these proteins along the pathway followed by the pollen tube during its journey to reach the egg and the central cell inside the embryo sac. AGP1 was expressed in the stigma, style, transmitting tract, and the chalazal and funiculus tissues of the ovules. AGP9 was present along the vasculature of the reproductive tissues and AGP12 was expressed in the stigmatic cells, chalazal and funiculus cells of the ovules, and in the septum. AGP15 was expressed in all pistil tissues, except in the transmitting tract, while AGP23 was specific to the pollen grain and pollen tube. The expression pattern of these AGPs provides new evidence for the detection of a subset of specific AGPs involved in plant reproductive processes, being of significance for this field of study. AGPs are prominent candidates for male–female communication during reproduction.This work was financed by FEDER through the COMPETE programme, and by Portuguese National funds through FCT – Fundação para a Ciência e Tecnologia (Project PTDC/AGR-GPL/115358/2009) and from an FCT PhD grant SFRH/BD/60995/2009 awarded to AMP. This project also benefited from financial support from the COST Action FA0903: ‘Harnessing Plant Reproduction for Crop Improvement’.Peer reviewe

Male-female communication triggers calcium signatures during fertilization in Arabidopsis

Cell-cell communication and interaction is critical during fertilization and triggers free cytosolic calcium ([Ca2+](cyto)) as a key signal for egg activation and a polyspermy block in animal oocytes. Fertilization in flowering plants is more complex, involving interaction of a pollen tube with egg adjoining synergid cells, culminating in release of two sperm cells and their fusion with the egg and central cell, respectively. Here, we report the occurrence and role of [Ca2+](cyto) signals during the entire double fertilization process in Arabidopsis. [Ca2+](cyto) oscillations are initiated in synergid cells after physical contact with the pollen tube apex. In egg and central cells, a short [Ca2+](cyto) transient is associated with pollen tube burst and sperm cell arrival. A second extended [Ca2+](cyto) transient solely in the egg cell is correlated with successful fertilization. Thus, each female cell type involved in double fertilization displays a characteristic [Ca2+](cyto) signature differing by timing and behaviour from [Ca2+](cyto) waves reported in mammals

Zygotic Genome Activation Occurs Shortly After Fertilization in Maize

The formation of a zygote via the fusion of an egg and sperm cell and its subsequent asymmetric division herald the start of the plant's life cycle. Zygotic genome activation (ZGA) is thought to occur gradually, with the initial steps of zygote and embryo development being primarily maternally controlled, and subsequent steps being governed by the zygotic genome. Here, using maize (Zea mays) as a model plant system, we determined the timing of zygote development and generated RNA-seq transcriptome profiles of gametes, zygotes, and apical and basal daughter cells. ZGA occurs shortly after fertilization and involves similar to 10% of the genome being activated in a highly dynamic pattern. In particular, genes encoding transcriptional regulators of various families are activated shortly after fertilization. Further analyses suggested that chromatin assembly is strongly modified after fertilization, that the egg cell is primed to activate the translational machinery, and that hormones likely play a minor role in the initial steps of early embryo development in maize. Our findings provide important insights into gamete and zygote activity in plants, and our RNA-seq transcriptome profiles represent a comprehensive, unique RNA-seq data set that can be used by the research community

Cell-Cell Communication in Plant Reproduction Let's get physical: gamete interaction in flowering plants

Abstract Fertilization comprises a series of precisely orchestrated steps that culminate in the fusion of male and female gametes. The most intimate steps during fertilization encompass gamete recognition, adhesion and fusion. In animals, some binding-effector proteins and enzymes have been identified that act on the cell surfaces of the gametes to regulate gamete compatibility and fertilization success. In contrast, exploring plant gamete interaction during double fertilization, a characteristic trait of flowering plants, has been hampered for a long time because of the protected location of the female gametes and technical limitations. Over the last couple of years, however, the use of advanced methodologies, new imaging tools and new mutants has provided deeper insights into double fertilization, at both the cellular and the molecular level, especially for the model plant Arabidopsis thaliana. Most likely, one consequence of inventing double fertilization may be the co-evolution of special molecular mechanisms to govern each successful sperm delivery and efficient gamete recognition and fusion. In vivo imaging of double fertilization and the recent discovery of numerous female-gametophyte-specific expressed genes encoding small secreted proteins, some of whom were found to be essential for the fertilization process, support this hypothesis. Nevertheless, recent findings indicate that at least the membrane-merger step in plant gamete interaction may rely on an ancient and widely used gamete fusion system

Twice the fun, double the trouble: gamete interactions in flowering plants

During sexual reproduction two gametes of opposite sex unite to produce a zygote. Gamete fusion is a highly controlled process and it has become evident that, across species, common concepts apply to this ancient and fundamental event. Sexual reproduction in flowering plants is even more complex in that two sperm cells fertilize two female reproductive cells (egg and central cell) in a process called double fertilization. Due to the coordinated developmental progression and mutual dependency of the two fertilization products (embryo and endosperm), the success and timing of the two fusion events substantially affects seed set. So far, four proteins are known to act on the surfaces of Arabidopsis gametes to accomplish double fertilization. The molecular and evolutionary characteristics of these players prove that flowering plants integrate plant-specific and widely conserved mechanisms to accomplish the timely fusion of each sperm cell with one female reproductive cell

Gamete fusion is facilitated by two sperm cell-expressed DUF679 membrane proteins

Successful double fertilization in flowering plants relies on two coordinated gamete fusion events, but the underlying molecular processes are not well understood. We show that two sperm-specific DOMAIN OF UNKNOWN FUNCTION 679 membrane proteins (DMP8 and DMP9) facilitate gamete fusion, with a greater effect on sperm-egg fusion than on sperm-central cell fusion. We also show that sperm adhesion and sperm cell separation depend on egg cell-secreted EGG CELL 1 proteins

F-Actin Organization and Pollen Tube Tip Growth in Arabidopsis Are Dependent on the Gametophyte-Specific Armadillo Repeat Protein ARO1[W]

The signal-mediated and spatially controlled assembly and dynamics of actin are crucial for maintaining shape, motility, and tip growth of eukaryotic cells. We report that a novel Armadillo repeat protein in Arabidopsis thaliana, ARMADILLO REPEAT ONLY1 (ARO1), is of fundamental importance for polar growth and F-actin organization in tip-growing pollen tubes. ARO1 is specifically expressed in the vegetative cell of pollen as well as in the egg cell. ARO1-GFP (for green fluorescent protein) fusion proteins accumulate most notably in pollen tube tips and partially colocalize with F-actin in the shank of pollen tubes. ARO1 knockout results in a highly disorganized actin cytoskeleton, growth depolarization, and ultimately tube growth arrest. Tip-localized ARO1-GFP is spatially shifted toward the future site of tip growth, indicating a role of ARO1 in the signaling network controlling tip growth and regulating actin organization. After the pollen tube discharges its contents into the receptive synergid, ARO1-GFP colocalizes with emerging F-actin structures near the site of sperm cell fusion, suggesting additional participation in the mechanism of sperm cell tracking toward the female gametes. The variable localization of ARO1 in the cytoplasm, the nucleus, and at the plasma membrane, however, indicates a multifunctional role like that of β-catenin/Armadillo and the p120 catenins

Knockin' on pollen's door: live cell imaging of early polarization events in germinating Arabidopsis pollen

Pollen tubes are an excellent system for studying the cellular dynamics and complex signaling pathways that coordinate polarized tip growth. Although several signaling mechanisms acting in the tip-growing pollen tube have been described, our knowledge on the subcellular and molecular events during pollen germination and growth site selection at the pollen plasma membrane is rather scarce. To simultaneously track germinating pollen from up to 12 genetically different plants we developed an inexpensive and easy mounting technique, suitable for every standard microscope setup. We performed high magnification live-cell imaging during Arabidopsis pollen activation, germination, and the establishment of pollen tube tip growth by using fluorescent marker lines labeling either the pollen cytoplasm, vesicles, the actin cytoskeleton or the sperm cell nuclei and membranes. Our studies revealed distinctive vesicle and F-actin polarization during pollen activation and characteristic growth kinetics during pollen germination and pollen tube formation. Initially, the germinating Arabidopsis pollen tube grows slowly and forms a uniform roundish bulge, followed by a transition phase with vesicles heavily accumulating at the growth site before switching to rapid tip growth. Furthermore, we found the two sperm cells to be transported into the pollen tube after the phase of rapid tip growth has been initiated. The method presented here is suitable to quantitatively study subcellular events during Arabidopsis pollen germination and growth, and for the detailed analysis of pollen mutants with respect to pollen polarization, bulging, or growth site selection at the pollen plasma membrane