Signalling between the sexes during pollen tube reception.

Plant reproduction is a complex, highly-coordinated process in which a single, male germ cell grows through the maternal reproductive tissues to reach and fertilise the egg cell. Focussing on Arabidopsis thaliana, we review signalling between male and female partners which is important throughout the pollen tube journey, especially during pollen tube reception at the ovule. Numerous receptor kinases and their coreceptors are implicated in signal perception in both the pollen tube and synergid cells at the ovule entrance, and several specific peptide and carbohydrate ligands for these receptors have recently been identified. Clarifying the interplay between these signals and the downstream responses they instigate presents a challenge for future research and may help to illuminate broader principles of plant cell-cell communication.

Redundant function of the Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K4-6 is essential for pollen germination.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] in eukaryotes. Although PIP5K genes are reported to be involved in pollen tube germination and growth, the essential roles of PIP5K in these processes remain unclear. Here, we performed a comprehensive genetic analysis of the Arabidopsis thaliana PIP5K4, PIP5K5, and PIP5K6 genes and revealed that their redundant function is essential for pollen germination. Pollen with the pip5k4pip5k5pip5k6 triple mutation was sterile, while pollen germination efficiency and pollen tube growth were reduced in the pip5k6 single mutant and further reduced in the pip5k4pip5k6 and pip5k5pip5k6 double mutants. YFP-fusion proteins, PIP5K4-YFP, PIP5K5-YFP, and PIP5K6-YFP, which could rescue the sterility of the triple mutant pollen, preferentially localized to the tricolpate aperture area and the future germination site on the plasma membrane prior to germination. Triple mutant pollen grains under the germination condition, in which spatiotemporal localization of the PtdIns(4,5)P2 fluorescent marker protein 2xmCHERRY-2xPHPLC as seen in the wild type was abolished, exhibited swelling and rupture of the pollen wall, but neither the conspicuous protruding site nor site-specific deposition of cell wall materials for germination. These data indicate that PIP5K4-6 and their product PtdIns(4,5)P2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain.

The RALF signaling pathway regulates cell wall integrity during pollen tube growth in maize.

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis (Arabidopsis thaliana). To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize (Zea mays). We show that Clade IB ZmRALF2/3 mutations, but not Clade III ZmRALF1/5 mutations, cause cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located in the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution patterns leading to altered cell wall organization and thickness culminating in pollen tube burst. Clade IB, but not Clade III ZmRALFs, strongly interact as ligands with the pollen-specific Catharanthus roseus RLK1-like (CrRLK1L) receptor kinases Z. mays FERONIA-like (ZmFERL) 4/7/9, LORELEI-like glycosylphosphatidylinositol-anchor (LLG) proteins Z. mays LLG 1 and 2 (ZmLLG1/2), and Z. mays pollen extension-like (PEX) cell wall proteins ZmPEX2/4. Notably, ZmFERL4 outcompetes ZmLLG2 and ZmPEX2 outcompetes ZmFERL4 for ZmRALF2 binding. Based on these data, we suggest that Clade IB RALFs act in a dual role as cell wall components and extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in maize and probably other plants.

Antagonistic RALF peptides control an intergeneric hybridization barrier on Brassicaceae stigmas.

Pollen-pistil interactions establish interspecific/intergeneric pre-zygotic hybridization barriers in plants. The rejection of undesired pollen at the stigma is crucial to avoid outcrossing but can be overcome with the support of mentor pollen. The mechanisms underlying this hybridization barrier are largely unknown. Here, in Arabidopsis, we demonstrate that receptor-like kinases FERONIA/CURVY1/ANJEA/HERCULES RECEPTOR KINASE 1 and cell wall proteins LRX3/4/5 interact on papilla cell surfaces with autocrine stigmatic RALF1/22/23/33 peptide ligands (sRALFs) to establish a lock that blocks the penetration of undesired pollen tubes. Compatible pollen-derived RALF10/11/12/13/25/26/30 peptides (pRALFs) act as a key, outcompeting sRALFs and enabling pollen tube penetration. By treating Arabidopsis stigmas with synthetic pRALFs, we unlock the barrier, facilitating pollen tube penetration from distantly related Brassicaceae species and resulting in interspecific/intergeneric hybrid embryo formation. Therefore, we uncover a "lock-and-key" system governing the hybridization breadth of interspecific/intergeneric crosses in Brassicaceae. Manipulating this system holds promise for facilitating broad hybridization in crops.

Insights into pollen-stigma recognition: self-incompatibility mechanisms serve as interspecies barriers in Brassicaceae?

A new study provides a comprehensive molecular mechanism that controls interspecific incompatibility of self-incompatible (SI) plants in the Brassicaceae. This finding points to a potentially promising path to break interspecific barriers and achieve introgression of desirable traits into crops from distant species among SI crops in the Brassicaceae.

Distinct chromatin signatures in the Arabidopsis male gametophyte.

Epigenetic reprogramming in the germline contributes to the erasure of epigenetic inheritance across generations in mammals but remains poorly characterized in plants. Here we profiled histone modifications throughout Arabidopsis male germline development. We find that the sperm cell has widespread apparent chromatin bivalency, which is established by the acquisition of H3K27me3 or H3K4me3 at pre-existing H3K4me3 or H3K27me3 regions, respectively. These bivalent domains are associated with a distinct transcriptional status. Somatic H3K27me3 is generally reduced in sperm, while dramatic loss of H3K27me3 is observed at only ~700 developmental genes. The incorporation of the histone variant H3.10 facilitates the establishment of sperm chromatin identity without a strong impact on resetting of somatic H3K27me3. Vegetative nuclei harbor thousands of specific H3K27me3 domains at repressed genes, while pollination-related genes are highly expressed and marked by gene body H3K4me3. Our work highlights putative chromatin bivalency and restricted resetting of H3K27me3 at developmental regulators as key features in plant pluripotent sperm.

Egg cell-secreted aspartic proteases ECS1/2 promote gamete attachment to prioritize the fertilization of egg cells over central cells in Arabidopsis.

Double fertilization is an innovative phenomenon in angiosperms, in which one sperm cell first fuses with the egg cell to produce the embryo, and then the other sperm fuses with the central cell to produce the endosperm. However, the molecular mechanism of the preferential fertilization of egg cells is poorly understood. In this study, we report that two egg cell-secreted aspartic proteases, ECS1 and ECS2, play an important role in promoting preferential fertilization of egg cells in Arabidopsis. We show that simultaneous loss of ECS1 and ECS2 function resulted in an approximately 20% reduction in fertility, which can be complemented by the full-length ECS1/2 but not by corresponding active site mutants or by secretion-defective versions of ECS1/2. Detailed phenotypic analysis revealed that the egg cell-sperm cell attachment was compromised in ecs1 ecs2 siliques. Limited pollination assays with cyclin-dependent kinase a1 (cdka;1) pollen showed that preferential egg cell fertilization was impaired in the ecs1 ecs2 mutant. Taken together, these results demonstrate that egg cells secret two aspartic proteases, ECS1 and ECS2, to facilitate the attachment of sperm cells to egg cells so that preferential fertilization of egg cells is achieved. This study reveals the molecular mechanism of preferential fertilization in Arabidopsis thaliana.

The egg cell is preferentially fertilized in Arabidopsis double fertilization.

In flowering plants (angiosperms), fertilization of the egg cell by one sperm cell produces an embryo, whereas fusion of a second sperm cell with the central cell generates the endosperm. In most angiosperms like Arabidopsis, a pollen grain contains two isomorphic sperm cells required for this double fertilization process. A long-standing unsolved question is whether the two fertilization events have any preference. A tool to address this question is the usage of the cyclin-dependent kinase a1 (cdka;1) mutant pollen, which produces a single sperm-like cell (SLC). Here, we first adopt a complementation-based fluorescence-labeling method to successfully separate and collect cdka;1 mutant pollen containing a single SLC. Single-cell RNA-sequencing analysis revealed that cdka;1 SLCs show a gene expression profile highly similar to that of sperm cells and not to the generative cell, precursor of the two sperm cells. Pollination assays using a limited number of cdka;1 mutant pollen revealed that in 98.2% of the ovules, single fertilization of the egg cell occurred. Pollination of pistils with excessive cdka;1 mutant pollen allowed the delivery of a second SLC via fertilization recovery, which fertilized the central cell, resulting in 20.7% double-fertilized ovules. This indicates that cdka;1 SLCs are able to fertilize both the egg and the central cell. Taken together, our findings have answered a long-standing question and support that preferential fertilization of the egg cell is evident in Arabidopsis.

Progressive chromatin silencing of ABA biosynthesis genes permits seed germination in Arabidopsis.

Seed germination represents a major developmental switch in plants that is vital to agriculture, but how this process is controlled at the chromatin level remains obscure. Here we demonstrate that successful germination in Arabidopsis thaliana requires a chromatin mechanism that progressively silences 9-CIS-EPOXYCAROTENOID DIOXYGENASE 6 (NCED6), which encodes a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, through the cooperative action of the RNA-binding protein RZ-1 and the polycomb repressive complex 2 (PRC2). Simultaneous inactivation of RZ-1 and PRC2 blocked germination and synergistically derepressed NCEDs and hundreds of genes. At NCED6, in part by promoting H3 deacetylation and suppressing H3K4me3, RZ-1 facilitates transcriptional silencing and also an H3K27me3 accumulation process that occurs during seed germination and early seedling growth. Genome-wide analysis revealed that RZ-1 is preferentially required for transcriptional silencing of many PRC2 targets early during seed germination, when H3K27me3 is not yet established. We propose RZ-1 confers a novel silencing mechanism to compensate for and synergize with PRC2. Our work highlights the progressive chromatin silencing of ABA biosynthesis genes via the RNA-binding protein RZ-1 and PRC2 acting in synergy, a process that is vital for seed germination.

RALF peptide signaling controls the polytubey block in Arabidopsis.

Fertilization of an egg by multiple sperm (polyspermy) leads to lethal genome imbalance and chromosome segregation defects. In Arabidopsis thaliana, the block to polyspermy is facilitated by a mechanism that prevents polytubey (the arrival of multiple pollen tubes to one ovule). We show here that FERONIA, ANJEA, and HERCULES RECEPTOR KINASE 1 receptor-like kinases located at the septum interact with pollen tube-specific RALF6, 7, 16, 36, and 37 peptide ligands to establish this polytubey block. The same combination of RALF (rapid alkalinization factor) peptides and receptor complexes controls pollen tube reception and rupture inside the targeted ovule. Pollen tube rupture releases the polytubey block at the septum, which allows the emergence of secondary pollen tubes upon fertilization failure. Thus, orchestrated steps in the fertilization process in Arabidopsis are coordinated by the same signaling components to guarantee and optimize reproductive success.

Lack of ethylene does not affect reproductive success and synergid cell death in Arabidopsis.

The signaling pathway of the gaseous hormone ethylene is involved in plant reproduction, growth, development, and stress responses. During reproduction, the two synergid cells of the angiosperm female gametophyte both undergo programmed cell death (PCD)/degeneration but in a different manner: PCD/degeneration of one synergid facilitates pollen tube rupture and thereby the release of sperm cells, while PCD/degeneration of the other synergid blocks supernumerary pollen tubes. Ethylene signaling was postulated to participate in some of the synergid cell functions, such as pollen tube attraction and the induction of PCD/degeneration. However, ethylene-mediated induction of synergid PCD/degeneration and the role of ethylene itself have not been firmly established. Here, we employed the CRISPR/Cas9 technology to knock out the five ethylene-biosynthesis 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes and created Arabidopsis mutants free of ethylene production. The ethylene-free mutant plants showed normal triple responses when treated with ethylene rather than 1-aminocyclopropane-1-carboxylic acid, but had increased lateral root density and enlarged petal sizes, which are typical phenotypes of mutants defective in ethylene signaling. Using these ethylene-free plants, we further demonstrated that production of ethylene is not necessarily required to trigger PCD/degeneration of the two synergid cells, but certain components of ethylene signaling including transcription factors ETHYLENE-INSENSITIVE 3 (EIN3) and EIN3-LIKE 1 (EIL1) are necessary for the death of the persistent synergid cell.

From birth to function: Male gametophyte development in flowering plants.

Male germline development in flowering plants involves two distinct and successive phases, microsporogenesis and microgametogenesis, which involve one meiosis followed by two rounds of mitosis. Many aspects of distinctions after mitosis between the vegetative cell and the male germ cells are seen, from morphology to structure, and the differential functions of the two cell types in the male gametophyte are differentially needed and required for double fertilization. The two sperm cells, carriers of the hereditary substances, depend on the vegetative cell/pollen tube to be delivered to the female gametophyte for double fertilization. Thus, the intercellular communication and coordinated activity within the male gametophyte probably represent the most subtle regulation in flowering plants to guarantee the success of reproduction. This review will focus on what we have known about the differentiation process and the functional diversification of the vegetative cell and the male germ cell, the most crucial cell types for plant fertility and crop production.

Stigmatic ROS: regulator of compatible pollen tube perception?

Accurate communication at the stigma surface is required to promote plants' own pollen and reject foreign pollen. Liu et al. have now discovered an autocrine signaling pathway at the surface of arabidopsis stigmatic papillae, accumulating ROS. Downregulation of ROS production via an antagonistic peptide from the pollen coat promotes pollen hydration and germination.

VPS18-regulated vesicle trafficking controls the secretion of pectin and its modifying enzyme during pollen tube growth in Arabidopsis.

In eukaryotes, homotypic fusion and vacuolar protein sorting (HOPS) as well as class C core vacuole/endosome tethering (CORVET) are evolutionarily conserved membrane tethering complexes that play important roles in lysosomal/vacuolar trafficking. Whether HOPS and CORVET control endomembrane trafficking in pollen tubes, the fastest growing plant cells, remains largely elusive. In this study, we demonstrate that the four core components shared by the two complexes, Vacuole protein sorting 11 (VPS11), VPS16, VPS33, and VPS18, are all essential for pollen tube growth in Arabidopsis thaliana and thus for plant reproduction success. We used VPS18 as a representative core component of the complexes to show that the protein is localized to both multivesicular bodies (MVBs) and the tonoplast in a growing pollen tube. Mutant vps18 pollen tubes grew more slowly in vivo, resulting in a significant reduction in male transmission efficiency. Additional studies revealed that membrane fusion from MVBs to vacuoles is severely compromised in vps18 pollen tubes, corroborating the function of VPS18 in late endocytic trafficking. Furthermore, vps18 pollen tubes produce excessive exocytic vesicles at the apical zone and excessive amounts of pectin and pectin methylesterases in the cell wall. In conclusion, this study establishes an additional conserved role of HOPS/CORVET in homotypic membrane fusion during vacuole biogenesis in pollen tubes and reveals a feedback regulation of HOPS/CORVET in the secretion of cell wall modification enzymes of rapidly growing plant cells.

AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance.

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

Semi-In Vivo Assay for Pollen Tube Attraction.

In flowering plants, each pollen tube delivers two sperm cells into the ovule to complete double fertilization. During the process, pollen tubes need to be navigated into the ovule, where accurate and complex pre-ovule guidance and ovule guidance are required. In recent years, different methods have been established to study those genes involved in the regulation of pollen tube guidance. Semi-in vivo ovule targeting mimics in vivo pollen tube micropylar guidance, and the semi-in vivo ovule targeting assay has been used to investigate function of genes involved in micropylar guidance. Moreover, the ovule targeting assay is the best way to do live cell imaging, which facilitates observation of pollen tube reception, synergid cell degeneration, and semi-in vivo gamete fusion. Meanwhile, semi-in vivo pollen tube attraction assay is another useful method to directly determine whether a certain molecule has pollen tube attraction activity.

Obtaining Mutant Pollen for Phenotypic Analysis and Pollen Tube Dual Staining.

Mutant phenotype observation is the most useful and important method to study which biological process a gene-of-interest is involved in. In flowering plants, excessive pollen grains land and germinate on the stigma, then pollen tubes grow through the transmitting tract to reach the ovules, eventually enter the micropyle to complete double fertilization. First, for mutants whose homozygotes could not be obtained due to pollen tube defects, it is difficult to observe the defect phenotype since the pollen grains of different genotypes are mixed together. Here, we provide a detailed protocol to pick out mutant pollen grains from the heterozygous mutant plants in Arabidopsis thaliana. By using this method, we could obtain sufficient mutant pollen grains for phenotypic analysis. Second, it is difficult to compare the pollen/pollen tube behavior of two different genotypes/species in vivo in a same pistil. Here, we develop a new dual staining method which combines GUS staining with aniline blue staining. By using this method, we can analyze the competence of the two different pollen tubes in the same pistil.