Mark A. Johnson

Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil.

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.

Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization.

In flowering plants, sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize an egg and a central cell. Here, we identified the gene encoding Arabidopsis HAP2, demonstrating that it is allelic to GCS1. HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to ovules. We identified an insertion (hap2-1) that disrupts the C-terminal portion of the protein and tags mutant pollen grains with the β-glucuronidase reporter. By monitoring reporter expression, we showed that hap2-1 does not diminish pollen tube length in vitro or in the pistil, but it reduces ovule targeting by twofold. In addition, we show that the hap2 sperm that are delivered to ovules fail to initiate fertilization. HAP2 is predicted to encode a protein with an N-terminal secretion signal, a single transmembrane domain and a C-terminal histidine-rich domain. These results point to a dual role for HAP2, functioning in both pollen tube guidance and in fertilization. Moreover, our findings suggest that sperm, long considered to be passive cargo, are involved in directing the pollen tube to its target.

Plotting a Course: Multiple Signals Guide Pollen Tubes to Their Targets

Pollen plays a critical role in the life cycle of all flowering plants, generating a polarized pollen tube that delivers sperm to the eggs in the interior of the flower. Pollen tubes perceive multiple extracellular signals during their extended growth through different floral environments; these environments discriminate among pollen grains, allowing only those that are appropriately recognized to invade. The phases of pollen tube growth include interactions that establish pollen polarity, entry of pollen tubes into female cell walls, and adhesion-based pollen tube motility through a carbohydrate-rich matrix. Recent studies have identified cells within the female germ unit as important sources of pollen guidance cues. Other signals undoubtedly exist, and their discovery will require genetic screens that target diploid tissues as well as haploid male and female cells.

Pollen Tubes Lacking a Pair of K + Transporters Fail to Target Ovules in Arabidopsis

How pollen tubes respond to female cues and precisely deliver sperm cells to the ovule is largely unknown. This article shows that two members of a cation transporter family are required in pollen tube navigation and in shifting polar tip growth. Flowering plant reproduction requires precise delivery of the sperm cells to the ovule by a pollen tube. Guidance signals from female cells are being identified; however, how pollen responds to those cues is largely unknown. Here, we show that two predicted cation/proton exchangers (CHX) in Arabidopsis thaliana, CHX21 and CHX23, are essential for pollen tube guidance. Male fertility was unchanged in single chx21 or chx23 mutants. However, fertility was impaired in chx21 chx23 double mutant pollen. Wild-type pistils pollinated with a limited number of single and double mutant pollen producing 62% fewer seeds than those pollinated with chx23 single mutant pollen, indicating that chx21 chx23 pollen is severely compromised. Double mutant pollen grains germinated and grew tubes down the transmitting tract, but the tubes failed to turn toward ovules. Furthermore, chx21 chx23 pollen tubes failed to enter the micropyle of excised ovules. Green fluorescent protein–tagged CHX23 driven by its native promoter was localized to the endoplasmic reticulum of pollen tubes. CHX23 mediated K+ transport, as CHX23 expression in Escherichia coli increased K+ uptake and growth in a pH-dependent manner. We propose that by modifying localized cation balance and pH, these transporters could affect steps in signal reception and/or transduction that are critical to shifting the axis of polarity and directing pollen growth toward the ovule.

Gamete fusion is required to block multiple pollen tubes from entering an Arabidopsis ovule

In double fertilization, a reproductive system unique to flowering plants, two immotile sperm are delivered to an ovule by a pollen tube. One sperm fuses with the egg to generate a zygote, the other with the central cell to produce endosperm. A mechanism preventing multiple pollen tubes from entering an ovule would ensure that only two sperm are delivered to female gametes. We use live-cell imaging and a novel mixed-pollination assay that can detect multiple pollen tubes and multiple sets of sperm within a single ovule to show that Arabidopsis efficiently prevents multiple pollen tubes from entering an ovule. However, when gamete-fusion defective hap2(gcs1) or duo1 sperm are delivered to ovules, as many as three additional pollen tubes are attracted. When gamete fusion fails, one of two pollen tube-attracting synergid cells persists, enabling the ovule to attract more pollen tubes for successful fertilization. This mechanism prevents the delivery of more than one pair of sperm to an ovule, provides a means of salvaging fertilization in ovules that have received defective sperm, and ensures maximum reproductive success by distributing pollen tubes to all ovules.

Is HAP2-GCS1 an ancestral gamete fusogen?

Eukaryotes employ a diversity of strategies to ensure that gametes come together, but the cellular climax is less varied: gamete plasma membranes must fuse to allow the combination of parental genomes. Recent studies of HAP2-GCS1, a sex-restricted transmembrane protein found in genomes representing all major eukaryotic taxa except fungi, suggest that a broad array of eukaryotic organisms could share a common mechanism for gamete fusion. Plant, protozoan, and algal gametes carrying loss-of-function mutations in HAP2-GCS1 fail to fuse with their complements. We propose that HAP2-GCS1 is a crucial component of an ancient mechanism that mediates the fusion of gamete plasma membranes and could have been a key early innovation in the evolution of sexual reproduction.

Neurons expressing trace amine-associated receptors project to discrete glomeruli and constitute an olfactory subsystem

Some chemoreceptors of the trace amine-associated receptor (TAAR) family detect innately aversive odors and are proposed to activate hardwired olfactory circuits. However, the wiring of TAAR neurons, the regulatory mechanisms of Taar gene choice, and the subcellular localization of TAAR proteins remain unknown. Here, we reveal similarities between neurons expressing TAARs and odorant receptors (ORs), but also unexpected differences. Like ORs, TAARs seem to be monoallelically expressed and localized both in cilia, the site of odor detection, and in axons, where they may participate in guidance. TAAR neurons project to discrete glomeruli predominantly localized to a confined bulb region. Taar expression involves different regulatory logic than OR expression, as neurons choosing a Taar5 knockout allele frequently express a second Taar without silencing the deleted allele. Moreover, the epigenetic signature of OR gene choice is absent from Taar genes. The unique molecular and anatomical features of the TAAR neurons suggest that they constitute a distinct olfactory subsystem.

Three MYB Transcription Factors Control Pollen Tube Differentiation Required for Sperm Release

In flowering plants, immotile sperm cells develop within the pollen grain and are delivered to female gametes by a pollen tube. Upon arrival at the female gametophyte, the pollen tube stops growing and releases sperm cells for successful fertilization. Several female signaling components essential for pollen tube reception have been identified; however, male components remain unknown. We show that the expression of three closely related MYB transcription factors is induced in pollen tubes by growth in the pistil. Pollen tubes lacking these three transcriptional regulators fail to stop growing in synergids, specialized cells flanking the egg cell that attract pollen tubes and degenerate upon pollen tube arrival. myb triple-mutant pollen tubes also fail to release their sperm cargo. We define a suite of pollen tube-expressed genes regulated by these critical MYBs and identify transporters, carbohydrate-active enzymes, and small peptides as candidate molecular mediators of pollen tube-female interactions necessary for flowering plant reproduction. Our data indicate that de novo transcription in the pollen tube nucleus during growth in the pistil leads to pollen tube differentiation required for release of sperm cells.

HAP2(GCS1)-Dependent Gamete Fusion Requires a Positively Charged Carboxy-Terminal Domain

HAP2(GCS1) is a deeply conserved sperm protein that is essential for gamete fusion. Here we use complementation assays to define major functional regions of the Arabidopsis thaliana ortholog using HAP2(GCS1) variants with modifications to regions amino(N) and carboxy(C) to its single transmembrane domain. These quantitative in vivo complementation studies show that the N-terminal region tolerates exchange with a closely related sequence, but not with a more distantly related plant sequence. In contrast, a distantly related C-terminus is functional in Arabidopsis, indicating that the primary sequence of the C-terminus is not critical. However, mutations that neutralized the charge of the C-terminus impair HAP2(GCS1)-dependent gamete fusion. Our results provide data identifying the essential functional features of this highly conserved sperm fusion protein. They suggest that the N-terminus functions by interacting with female gamete-expressed proteins and that the positively charged C-terminus may function through electrostatic interactions with the sperm plasma membrane.

Expressing the Diphtheria Toxin A Subunit from the HAP2(GCS1) Promoter Blocks Sperm Maturation and Produces Single Sperm-Like Cells Capable of Fertilization

After meiosis, the male germline of flowering plants undergoes two mitoses, producing two sperm that are carried within a pollen tube to an ovule. One sperm fuses with the egg to form the zygote and the other fuses with the central cell to form the primary endosperm. The mechanisms that control male germline development and gene expression, and ensure that sperm properly fuse with female gametes are just beginning to be understood. Expression of the potent translation inhibitor, diphtheria toxin A subunit, from the Arabidopsis (Arabidopsis thaliana) HAP2(GCS1) promoter blocked sperm development before the final cell division, resulting in pollen tubes that carried a single sperm-like cell rather than two sperm. These pollen tubes targeted ovules and fertilized either the egg or the central cell, producing seeds with either endosperm or an embryo, but not both. Endosperm-only seeds significantly outnumbered embryo-only seeds, suggesting that single sperm-like cells preferentially fuse with the central cell. These experiments show that de novo translation is required for completion of sperm development, that the HAP2(GCS1) promoter is very tightly controlled, and that disruption of gene expression can result in male germ cells with a bias for gamete fusion.