Pollen Tube Reception Research Articles

Arabidopsis thaliana MLO genes are expressed in discrete domains during reproductive development.

MLOs in Plant Reproduction. The MILDEW RESISTANCE LOCUS-O (MLO) protein family, comprised of 15 members, plays roles in diverse cell-cell communication processes such as powdery mildew susceptibility, root thigmomorphogenesis, and pollen tube reception. The NORTIA (NTA, AtMLO7) gene is expressed in the synergid cells of the female gametophyte where it functions in intercellular communication with the pollen tube. Discrepancies between previously published promoter::GUS and promoter::gene-GUS constructs expression patterns led us to explore the regulation of NTA expression. Here we found via NTApro::gNTA-GUS truncations that sequences within the NTA gene negatively regulate its expression in the stomata and carpel walls. This led to the hypothesis that other MLO family members may also have additional regulatory sequences within the gene. MLOpro::gMLO-GUS constructs were examined for each family member focusing specifically on flowers in order to determine whether other MLOs could play a role in reproductive cell-cell communication. Notably, several MLOs were expressed in the pollen, in the stigma, in the pollinated style, and in the synergids and central cell. These findings indicate that other MLOs in addition to NTA could play a role in reproduction. Previous studies on the MLO family showed that phylogenetically related MLOs had redundant functions in powdery mildew infection and root thigmomorphogenesis; however, MLO expression in reproductive tissues did not strictly follow phylogenetic relationships, indicating that MLOs from different evolutionary origins may have been recruited for function in sexual reproduction.

Cell type-dependent localization of MLO proteins

ABSTRACTMildew resistance locus O (MLO) proteins are transmembrane proteins that mediate cell-cell communication in plants. We recently demonstrated the importance of subcellular localization to MLO function during pollen tube reception. NORTIA (NTA), the MLO protein involved in this process, localizes to the Golgi of the synergid cell before interaction with the pollen tube. MLO proteins that can substitute for NTA's function in this pathway all partially localize with the same Golgi marker in the synergid cell. In this study, we report that MLO subcellular localization is cell type-dependent, with different distributions of some MLOs observed when ectopically expressed in the epidermal cells of tobacco and Arabidopsis compared to synergids. This dependency may be due to co-factors that influence MLO function within a given cell type, providing an intriguing new target for understanding MLO distribution and subsequent function in their respective processes.

Arabidopsis LORELEI, a Maternally Expressed Imprinted Gene, Promotes Early Seed Development.

In flowering plants, the female gametophyte controls pollen tube reception immediately before fertilization and regulates seed development immediately after fertilization, although the controlling mechanisms remain poorly understood. Previously, we showed that LORELEI (LRE), which encodes a putative glycosylphosphatidylinositol-anchored membrane protein, is critical for pollen tube reception by the female gametophyte before fertilization and the initiation of seed development after fertilization. Here, we show that LRE is expressed in the synergid, egg, and central cells of the female gametophyte and in the zygote and proliferating endosperm of the Arabidopsis (Arabidopsis thaliana) seed. Interestingly, LRE expression in the developing seeds was primarily from the matrigenic LRE allele, indicating that LRE expression is imprinted. However, LRE was biallelically expressed in 8-d-old seedlings, indicating that the patrigenic allele does not remain silenced throughout the sporophytic generation. Regulation of imprinted LRE expression is likely novel, as LRE was not expressed in pollen or pollen tubes of mutants defective for MET1, DDM1, RNA-dependent DNA methylation, or MSI-dependent histone methylation. Additionally, the patrigenic LRE allele inherited from these mutants was not expressed in seeds. Surprisingly, and contrary to the predictions of the parental conflict hypothesis, LRE promotes growth in seeds, as loss of the matrigenic but not the patrigenic LRE allele caused delayed initiation of seed development. Our results showed that LRE is a rare imprinted gene that functions immediately after double fertilization and supported the model that a passage through the female gametophyte establishes monoalleleic expression of LRE in seeds and controls early seed development.

MILDEW RESISTANCE LOCUS O Function in Pollen Tube Reception Is Linked to Its Oligomerization and Subcellular Distribution.

Sexual reproduction in flowering plants requires communication between synergid cells and a tip-elongating pollen tube (PT) for the successful delivery of sperm cells to the embryo sac. The reception of the PT relies on signaling within the synergid cell that ultimately leads to the degeneration of the receptive synergid and PT rupture, releasing the sperm cells for double fertilization. In Arabidopsis (Arabidopsis thaliana), NORTIA, a member of the MILDEW RESISTANCE LOCUS O (MLO) family of proteins, plays a critical role in the communication processes regulating PT reception. In this study, we determined that MLO function in PT reception is dependent on MLO protein localization into a Golgi-associated compartment before PT arrival, indicating that PT-triggered regulation of the synergid secretory system is important for synergid function during pollination. Additionally, a structure-function analysis revealed that MLO homooligomerization, mediated by the amino-terminal region of the protein, and carboxyl-terminal tail identity both contribute to MLO activity during PT reception.

Arabidopsis adaptor protein 1G is critical for pollen development

Pollen development is a pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 (AP1G) mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development.

AP1G mediates vacuolar acidification during synergid-controlled pollen tube reception

Double fertilization in angiosperms requires the delivery of immotile sperm through pollen tubes, which enter embryo sacs to initiate synergid degeneration and to discharge. This fascinating process, called pollen tube reception, involves extensive communications between pollen tubes and synergids, within which few intracellular regulators involved have been revealed. Here, we report that vacuolar acidification in synergids mediated by AP1G and V-ATPases might be critical for pollen tube reception. Functional loss of AP1G or VHA-A, encoding the γ subunit of adaptor protein 1 or the shared component of two endomembrane V-ATPases, respectively, impaired synergid-controlled pollen tube reception and caused partial female sterility. AP1G works in parallel to the plasma membrane-associated receptor FERONIA in synergids, suggesting that synergid-mediated pollen tube reception requires proper sorting of vacuolar cargos by AP1G. Although AP1G did not mediate the targeting of V-ATPases, AP1G loss of function or the expression of AP1G-RNAi compromised vacuolar acidification mediated by V-ATPases, implying their genetic interaction. We propose that vacuolar acidification might represent a distinct cell-death mechanism specifically adopted by the plant phylum, which is critical for synergid degeneration during pollen tube reception.

Maternal ENODLs Are Required for Pollen Tube Reception in Arabidopsis

During the angiosperm (flowering-plant) life cycle, double fertilization represents the hallmark between diploid and haploid generations [1]. The success of double fertilization largely depends on compatible communication between the male gametophyte (pollen tube) and the maternal tissues of the flower, culminating in precise pollen tube guidance to the female gametophyte (embryo sac) and its rupture torelease sperm cells. Several important factors involved in the pollen tube reception have been identified recently [2-6], but the underlying signaling pathways are far from being understood. Here, we report that a group of female-specific small proteins, early nodulin-like proteins (ENODLs, or ENs), are required for pollen tube reception. ENs are featured with a plastocyanin-like (PCNL) domain, an arabinogalactan (AG) glycomodule, and a predicted glycosylphosphatidylinositol (GPI) anchor motif. We show that ENs are asymmetrically distributed at the plasma membrane of the synergid cells and accumulate at the filiform apparatus, where arriving pollen tubes communicate with the embryo sac. EN14 strongly and specifically interacts with the extracellular domain of the receptor-like kinase FERONIA, localized at the synergid cell surface and known to critically control pollen tube reception [6]. Wild-type pollen tubes failed to arrest growth and to rupture after entering the ovules of quintuple loss-of-function EN mutants, indicating a central role of ENs in male-female communication and pollen tube reception. Moreover, overexpression of EN15 by the endogenous promoter caused disturbed pollen tube guidance and reduced fertility. These data suggest that female-derived GPI-anchored ENODLs play an essential role in male-female communication and fertilization.

The Role of LORELEI in Pollen Tube Reception at the Interface of the Synergid Cell and Pollen Tube Requires the Modified Eight-Cysteine Motif and the Receptor-Like Kinase FERONIA.

In angiosperms, pollen tube reception by the female gametophyte is required for sperm release and double fertilization. In Arabidopsis thaliana lorelei (lre) mutants, pollen tube reception fails in most female gametophytes, which thus remain unfertilized. LRE encodes a putative glycosylphosphatidylinositol (GPI)-anchored surface protein with a modified eight-cysteine motif (M8CM). LRE fused to citrine yellow fluorescent protein (LRE-cYFP) remains functional and localizes to the synergid plasma membrane-rich filiform apparatus, the first point of contact between the pollen tube and the female gametophyte. Structure-function analysis using LRE-cYFP showed that the role of LRE in pollen tube reception requires the M8CM, but not the domains required for GPI anchor addition. Consistently, LRE-cYFP-TM, where GPI anchor addition domains were replaced with a single-pass transmembrane domain, fully complemented the pollen tube reception defect in lre-7 female gametophytes. Ectopically expressed and delivered LRE-cYFP from pollen tubes could non-cell-autonomously complement the pollen tube reception defect in lre female gametophytes, only if they expressed FERONIA. Additionally, pollen tube-expressing LRE variants lacking domains critical for GPI anchor addition also rescued lre female gametophyte function. Therefore, LRE and FERONIA jointly function in pollen tube reception at the interface of the synergid cell and pollen tube.

Genome-Wide Study of the Tomato SlMLO Gene Family and Its Functional Characterization in Response to the Powdery Mildew Fungus Oidium neolycopersici.

The MLO (Mildew Locus O) gene family encodes plant-specific proteins containing seven transmembrane domains and likely acting in signal transduction in a calcium and calmodulin dependent manner. Some members of the MLO family are susceptibility factors toward fungi causing the powdery mildew disease. In tomato, for example, the loss-of-function of the MLO gene SlMLO1 leads to a particular form of powdery mildew resistance, called ol-2, which arrests almost completely fungal penetration. This type of penetration resistance is characterized by the apposition of papillae at the sites of plant-pathogen interaction. Other MLO homologs in Arabidopsis regulate root response to mechanical stimuli (AtMLO4 and AtMLO11) and pollen tube reception by the female gametophyte (AtMLO7). However, the role of most MLO genes remains unknown. In this work, we provide a genome-wide study of the tomato SlMLO gene family. Besides SlMLO1, other 15 SlMLO homologs were identified and characterized with respect to their structure, genomic organization, phylogenetic relationship, and expression profile. In addition, by analysis of transgenic plants, we demonstrated that simultaneous silencing of SlMLO1 and two of its closely related homologs, SlMLO5 and SlMLO8, confer higher level of resistance than the one associated with the ol-2 mutation. The outcome of this study provides evidence for functional redundancy among tomato homolog genes involved in powdery mildew susceptibility. Moreover, we developed a series of transgenic lines silenced for individual SlMLO homologs, which lay the foundation for further investigations aimed at assigning new biological functions to the MLO gene family.

A subunit of the oligosaccharyltransferase complex is required for interspecific gametophyte recognition in Arabidopsis.

Species-specific gamete recognition is a key premise to ensure reproductive success and the maintenance of species boundaries. During plant pollen tube (PT) reception, gametophyte interactions likely allow the species-specific recognition of signals from the PT (male gametophyte) by the embryo sac (female gametophyte), resulting in PT rupture, sperm release, and double fertilization. This process is impaired in interspecific crosses between Arabidopsis thaliana and related species, leading to PT overgrowth and a failure to deliver the sperm cells. Here we show that ARTUMES (ARU) specifically regulates the recognition of interspecific PTs in A. thaliana. ARU, identified in a genome-wide association study (GWAS), exclusively influences interspecific—but not intraspecific—gametophyte interactions. ARU encodes the OST3/6 subunit of the oligosaccharyltransferase complex conferring protein N-glycosylation. Our results suggest that glycosylation patterns of cell surface proteins may represent an important mechanism of gametophyte recognition and thus speciation.

SUN anchors pollen WIP-WIT complexes at the vegetative nuclear envelope and is necessary for pollen tube targeting and fertility.

LINC (linker of nucleoskeleton and cytoskeleton) complexes play an essential role in nuclear migration by connecting the nucleus to the cytoskeleton and/or motor proteins. Plant LINC complexes have recently been identified in Arabidopsis thaliana, with the inner nuclear membrane SUN and outer nuclear membrane WIP proteins comprising the first identified complex. A recent study identified a nuclear movement defect in Arabidopsis pollen vegetative nuclei linked to the outer nuclear envelope WIP and WIT proteins. However, the role that SUN proteins may play in pollen nuclear migration has yet to be addressed. To explore this question, a SUN2 lumenal domain that was targeted to the ER specifically in pollen was over-expressed. It is shown that the ER-targeted SUN2 lumenal domain was able to displace WIP and WIT proteins from the pollen vegetative nuclear envelope. Expression of this dominant-negative transgene led to impaired VN mobility, impaired pollen tube guidance, and defective pollen tube reception. The observed pollen defects are similar to phenotypes observed in a wip1-1 wip2-1 wip3-1 wit1-1 wit2-1 mutant. It is also shown that these defects were dependent on the KASH-binding function of the SUN2 lumenal domain. These data support a model where LINC complexes formed by SUN, WIP, and WIT at the VNE are responsible for VN migration and suggest an important function of SUN, WIP, and WIT in pollen tube guidance and reception.

Pollen Tube Discharge Completes the Process of Synergid Degeneration That Is Initiated by Pollen Tube-Synergid Interaction in Arabidopsis

In flowering plant reproduction, pollen tube reception is the signaling system that results in pollen tube discharge, synergid degeneration, and successful delivery of male gametes (two sperm cells) to the site where they can fuse with female gametes (egg cell and central cell). Some molecules required for this complex and essential signaling exchange have been identified; however, fundamental questions about the nature of the interactions between the pollen tube and the synergid cells remain to be clarified. Here, we monitor pollen tube arrival, pollen tube discharge, and synergid degeneration in Arabidopsis (Arabidopsis thaliana) wild type and in male and female gametophytic mutants that disrupt development and function of the gametophytes. By combining assays used previously to study these interactions and an assay that facilitates simultaneous analysis of pollen tube discharge and synergid degeneration, we find that synergid degeneration could be initiated without pollen tube discharge. Our data support the hypothesis that pollen tube-synergid contact, or signaling via secreted molecules, initiates receptive synergid degeneration. We also find that when pollen tubes successfully burst, they always discharge into a degenerated synergid. In addition to this pollen tube-dependent promotion of synergid degeneration, we also show that a basal developmental pathway mediates synergid degeneration in the absence of pollination. Our results are consistent with the model that a complex set of interactions between the pollen tube and synergid cells promote receptive synergid degeneration.

TURAN and EVAN Mediate Pollen Tube Reception in Arabidopsis Synergids through Protein Glycosylation

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.

Functional analysis of related CrRLK1L receptor-like kinases in pollen tube reception.

The Catharanthus roseus Receptor-Like Kinase 1-like (CrRLK1L) family of 17 receptor-like kinases (RLKs) has been implicated in a variety of signaling pathways in Arabidopsis, ranging from pollen tube (PT) reception and tip growth to hormonal responses. The extracellular domains of these RLKs have malectin-like domains predicted to bind carbohydrate moieties. Domain swap analysis showed that the extracellular domains of the three members analyzed (FER, ANX1, HERK1) are not interchangeable, suggesting distinct upstream components, such as ligands and/or co-factors. In contrast, their intercellular domains are functionally equivalent for PT reception, indicating that they have common downstream targets in their signaling pathways. The kinase domain is necessary for FER function, but kinase activity itself is not, indicating that other kinases may be involved in signal transduction during PT reception.

A Calcium Dialog Mediated by the FERONIA Signal Transduction Pathway Controls Plant Sperm Delivery

Sperm delivery for double fertilization of flowering plants relies on interactions between the pollen tube (PT) and two synergids, leading to programmed cell death (PCD) of the PT and one synergid. The mechanisms underlying the communication among these cells during PT reception is unknown. We discovered that the synergids control this process by coordinating their distinct calcium signatures in response to the calcium dynamics and growth behavior of the PT. Induced and spontaneous aberrant calcium responses in the synergids abolish the two coordinated PCD events. Components of the FERONIA (FER) signaling pathway are required for initiating and modulating these calcium responses and for coupling the PCD events. Intriguingly, the calcium signatures are interchangeable between the two synergids, implying that their fates of death and survival are determined by reversible interactions with the PT. Thus, complex intercellular interactions involving a receptor kinase pathway and calcium-mediated signaling control sperm delivery in plants.

Interactions between pollen tube and pistil control pollen tube identity and sperm release in the Arabidopsis female gametophyte

Flowering plants have immotile sperm that develop within the pollen cytoplasm and are delivered to female gametes by a pollen tube, a highly polarized extension of the pollen cell. In many flowering plant species, including seed crop plants, hundreds of pollen tubes grow towards a limited number of ovules. This system should ensure maximal fertilization of ovules and seed production; however, we know very little about how signalling between the critical cells is integrated to orchestrate delivery of two functional sperm to each ovule. Recent studies suggest that the pollen tube changes its gene-expression programme in response to growth through pistil tissue and that this differentiation process is critical for pollen tube attraction by the female gametophyte and for release of sperm. Interestingly, these two signalling systems, called pollen tube guidance and pollen tube reception, are also species-preferential. The present review focuses on Arabidopsis pollen tube differentiation within the pistil and addresses the idea that pollen tube differentiation defines pollen tube identity and recognition by female cells. We review recent identification of genes that may control pollen tube-female gametophyte recognition and discuss how these may be involved in blocking interspecific hybridization.

MYB97, MYB101 and MYB120 Function as Male Factors That Control Pollen Tube-Synergid Interaction in Arabidopsis thaliana Fertilization

Pollen tube reception involves a pollen tube-synergid interaction that controls the discharge of sperm cells into the embryo sac during plant fertilization. Despite its importance in the sexual reproduction of plants, little is known about the role of gene regulation in this process. We report here that the pollen-expressed transcription factors MYB97, MYB101 and MYB120 probably control genes whose encoded proteins play important roles in Arabidopsis thaliana pollen tube reception. They share a high amino acid sequence identity and are expressed mainly in mature pollen grains and pollen tubes. None of the single or double mutants of these three genes exhibited any visible defective phenotype. Although the myb97 myb101 myb120 triple mutant was not defective in pollen development, pollen germination, pollen tube growth or tube guidance, the pollen tubes of the triple mutants exhibited uncontrolled growth and failed to discharge their sperm cells after entering the embryo sac. In addition, the myb97 myb101 myb120 triple mutation significantly affected the expression of a group of pollen-expressed genes in mature pollen grains. All these results indicate that MYB97, MYB101 and MYB120 participate in pollen tube reception, possibly by controlling the expression of downstream genes.

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.

Conserved Molecular Components for Pollen Tube Reception and Fungal Invasion

During sexual reproduction in flowering plants such as Arabidopsis, a tip-growing pollen tube (PT) is guided to the synergid cells of the female gametophyte, where it bursts and releases the two sperm. Here we show that PT reception and powdery mildew (PM) infection, which involves communication between a tip-growing hypha and a plant epidermal cell, share molecular components. NORTIA (NTA), a member of the MLO family originally discovered in the context of PM resistance, and FERONIA (FER), a receptor-like kinase, both control PT reception in synergids. Homozygous fer mutants also display PM resistance, revealing a new function for FER and suggesting that conserved components, such as FER and distinct MLO proteins, are involved in both PT reception and PM infection.

FERONIA Is a Key Modulator of Brassinosteroid and Ethylene Responsiveness in Arabidopsis Hypocotyls

Ethylene signaling is a complex pathway that has been intensively analyzed partly due to its importance to the manifestation of horticultural phenomena, including fruit ripening and tissue senescence. In order to further our understanding of how this pathway is regulated, a screen for Arabidopsis mutants with increased ethylene response was conducted. From this, a mutant was identified as having a dark-grown hypocotyl that is indistinguishable from Col-0 wt in the presence of the ethylene perception inhibitor AgNO₃, yet has extreme responsiveness to even low levels of ethylene. Map-based cloning of the mutation revealed a T-DNA insertion in the coding sequence of the receptor-like kinase FERONIA, which is required for normal pollen tube reception and cell elongation in a currently unknown capacity. In contrast to a previous report, analysis of our feronia knockout mutant shows it also has altered responsiveness to brassinosteroids, with etiolated fer-2 seedlings being partially brassinosteroid insensitive with regard to promotion of hypocotyl elongation. Our results indicate that FERONIA-dependent brassinosteroid response serves to antagonize the effect of ethylene on hypocotyl growth of etiolated seedlings, with loss of proper brassinosteroid signaling disrupting this balance and leading to a greater impact of ethylene on hypocotyl shortening.