A wave of specific transcript and protein accumulation accompanies pollen dehydration.

In flowering plants, male gametes are immotile and carried by dry pollen grains to the female organ. Dehydrated pollen is thought to withstand abiotic stress when grains are dispersed from the anther to the pistil, after which sperm cells are delivered via pollen tube growth for fertilization and seed set. Yet, the underlying molecular changes accompanying dehydration and the impact on pollen development are poorly understood. To gain a systems perspective, we analyzed published transcriptomes and proteomes of developing Arabidopsis thaliana pollen. Waves of transcripts are evident as microspores develop to bicellular, tricellular, and mature pollen. Between the "early"- and "late"-pollen-expressed genes, an unrecognized cluster of transcripts accumulated, including those encoding late-embryogenesis abundant (LEA), desiccation-related protein, transporters, lipid-droplet associated proteins, pectin modifiers, cysteine-rich proteins, and mRNA-binding proteins. Results suggest dehydration onset initiates after bicellular pollen is formed. Proteins accumulating in mature pollen like ribosomal proteins, initiation factors, and chaperones are likely components of mRNA-protein condensates resembling "stress" granules. Our analysis has revealed many new transcripts and proteins that accompany dehydration in developing pollen. Together with published functional studies, our results point to multiple processes, including (1) protect developing pollen from hyperosmotic stress, (2) remodel the endomembrane system and walls, (3) maintain energy metabolism, (4) stabilize presynthesized mRNA and proteins in condensates of dry pollen, and (5) equip pollen for compatibility determination at the stigma and for recovery at rehydration. These findings offer novel models and molecular candidates to further determine the mechanistic basis of dehydration and desiccation tolerance in plants.

JAGGER localization and function are dependent on GPI anchor addition.

KEY MESSAGE: GPI anchor addition is important for JAGGER localization and in vivo function. Loss of correct GPI anchor addition in JAGGER, negatively affects its localization and function. In flowering plants, successful double fertilization requires the correct delivery of two sperm cells to the female gametophyte inside the ovule. The delivery of a single pair of sperm cells is achieved by the entrance of a single pollen tube into one female gametophyte. To prevent polyspermy, Arabidopsis ovules avoid the attraction of multiple pollen tubes to one ovule-polytubey block. In Arabidopsis jagger mutants, a significant number of ovules attract more than one pollen tube to an ovule due to an impairment in synergid degeneration. JAGGER encodes a putative arabinogalactan protein which is predicted to be anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Here, we show that JAGGER fused to citrine yellow fluorescent protein (JAGGER-cYFP) is functional and localizes mostly to the periphery of ovule integuments and transmitting tract cells. We further investigated the importance of GPI-anchor addition domains for JAGGER localization and function. Different JAGGER proteins with deletions in predicted ω-site regions and GPI attachment signal domain, expected to compromise the addition of the GPI anchor, led to disruption of JAGGER localization in the cell periphery. All JAGGER proteins with disrupted localization were also not able to rescue the polytubey phenotype, pointing to the importance of GPI-anchor addition to in vivo function of the JAGGER protein.

Epigenetic regulation of female germline development through ERECTA signaling pathway.

Germline development is a key step in sexual reproduction. Sexual plant reproduction begins with the formation of haploid spores by meiosis of megaspore mother cells (MMCs). Although many evidences, directly or indirectly, show that epigenetics plays an important role in MMC specification, how it controls the commitment of the MMC to downstream stages of germline development is still unclear. Electrophoretic mobility shift assay (EMSA), western blot, immunofluorescence, and chromatin immunoprecipitation coupled with quantitative PCR analyses were performed. Genetic interactions between BZR1 transcription factor family and the SWR1-SDG2-ER pathway in the control of female germline development were further studied. The present findings showed in Arabidopsis that two epigenetic factors, the chromatin remodeling complex SWI2/SNF2-RELATED 1 (SWR1) and a writer for H3K4me3 histone modification SET DOMAIN GROUP 2 (SDG2), genetically interact with the ERECTA (ER) receptor kinase signaling pathway and regulate female germline development by restricting the MMC cell fate to a single cell in the ovule primordium and ensure that only that single cell undergoes meiosis and subsequent megaspore degeneration. We also showed that SWR1-SDG2-ER signaling module regulates female germline development by promoting the protein accumulation of BZR1 transcription factor family on the promoters of primary miRNA processing factors, HYPONASTIC LEAVES 1 (HYL1), DICER-LIKE 1 (DCL1), and SERRATE (SE) to activate their expression. Our study elucidated a Gene Regulation Network that provides new insights for understanding how epigenetic factors and receptor kinase signaling pathways function in concert to control female germline development in Arabidopsis.

Stigma receptors control intraspecies and interspecies barriers in Brassicaceae.

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4-6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7-9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12-14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

Evolutionary analysis of the LORELEI gene family in plants reveals regulatory subfunctionalization.

A signaling complex comprising members of the LORELEI (LRE)-LIKE GPI-anchored protein (LLG) and Catharanthus roseus RECEPTOR-LIKE KINASE 1-LIKE (CrRLK1L) families perceive RAPID ALKALINIZATION FACTOR (RALF) peptides and regulate growth, reproduction, immunity, and stress responses in Arabidopsis (Arabidopsis thaliana). Genes encoding these proteins are members of multigene families in most angiosperms and could generate thousands of signaling complex variants. However, the links between expansion of these gene families and the functional diversification of this critical signaling complex as well as the evolutionary factors underlying the maintenance of gene duplicates remain unknown. Here, we investigated LLG gene family evolution by sampling land plant genomes and explored the function and expression of angiosperm LLGs. We found that LLG diversity within major land plant lineages is primarily due to lineage-specific duplication events, and that these duplications occurred both early in the history of these lineages and more recently. Our complementation and expression analyses showed that expression divergence (i.e. regulatory subfunctionalization), rather than functional divergence, explains the retention of LLG paralogs. Interestingly, all but one monocot and all eudicot species examined had an LLG copy with preferential expression in male reproductive tissues, while the other duplicate copies showed highest levels of expression in female or vegetative tissues. The single LLG copy in Amborella trichopoda is expressed vastly higher in male compared to in female reproductive or vegetative tissues. We propose that expression divergence plays an important role in retention of LLG duplicates in angiosperms.

The SEEL motif and members of the MYB-related REVEILLE transcription factor family are important for the expression of LORELEI in the synergid cells of the Arabidopsis female gametophyte.

Synergid cells in the micropylar end of the female gametophyte are required for critical cell-cell signaling interactions between the pollen tube and the ovule that precede double fertilization and seed formation in flowering plants. LORELEI (LRE) encodes a putative GPI-anchored protein that is expressed primarily in the synergid cells, and together with FERONIA, a receptor-like kinase, it controls pollen tube reception by the receptive synergid cell. Still, how LRE expression is controlled in synergid cells remains poorly characterized. We identified candidate cis-regulatory elements enriched in LRE and other synergid cell-expressed genes. One of the candidate motifs ('TAATATCT') in the LRE promoter was an uncharacterized variant of the Evening Element motif that we named as the Short Evening Element-like (SEEL) motif. Deletion or point mutations in the SEEL motif of the LRE promoter resulted in decreased reporter expression in synergid cells, demonstrating that the SEEL motif is important for expression of LRE in synergid cells. Additionally, we found that LRE expression is decreased in the loss of function mutants of REVEILLE (RVE) transcription factors, which are clock genes known to bind the SEEL and other closely related motifs. We propose that RVE transcription factors regulate LRE expression in synergid cells by binding to the SEEL motif in the LRE promoter. Identification of cis-regulatory elements and transcription factors involved in the expression of LRE will serve as a foundation to characterize the gene regulatory networks in synergid cells.

Holistic insights from pollen omics: co-opting stress-responsive genes and ER-mediated proteostasis for male fertility.

Sexual reproduction in flowering plants takes place without an aqueous environment. Sperm are carried by pollen through air to reach the female gametophyte, though the molecular basis underlying the protective strategy of the male gametophyte is poorly understood. Here we compared the published transcriptomes of Arabidopsis thaliana pollen, and of heat-responsive genes, and uncovered insights into how mature pollen (MP) tolerates desiccation, while developing and germinating pollen are vulnerable to heat stress. Germinating pollen expresses molecular chaperones or "heat shock proteins" in the absence of heat stress. Furthermore, pollen tubes that grew through pistils at basal temperature showed induction of the endoplasmic reticulum (ER) stress response, which is a characteristic of stressed vegetative tissues. Recent studies show MP contains mRNA-protein (mRNP) aggregates that resemble "stress" granules triggered by heat or other stresses to protect cells. Based on these observations, we postulate that mRNP particles are formed in maturing pollen in response to developmentally programmed dehydration. Dry pollen can withstand harsh conditions as it is dispersed in air. We propose that, when pollen lands on a compatible pistil and hydrates, mRNAs stored in particles are released, aided by molecular chaperones, to become translationally active. Pollen responds to osmotic, mechanical, oxidative, and peptide cues that promote ER-mediated proteostasis and membrane trafficking for tube growth and sperm discharge. Unlike vegetative tissues, pollen depends on stress-protection strategies for its normal development and function. Thus, heat stress during reproduction likely triggers changes that interfere with the normal pollen responses, thereby compromising male fertility. This holistic perspective provides a framework to understand the basis of heat-tolerant strains in the reproduction of crops.

Spatiotemporal control of miR398 biogenesis, via chromatin remodeling and kinase signaling, ensures proper ovule development.

The coordinated development of sporophytic and gametophytic tissues is essential for proper ovule patterning and fertility. However, the mechanisms regulating their integrated development remain poorly understood. Here, we report that the Swi2/Snf2-Related1 (SWR1) chromatin-remodeling complex acts with the ERECTA receptor kinase-signaling pathway to control female gametophyte and integument growth in Arabidopsis thaliana by inhibiting transcription of the microRNA gene MIR398c in early-stage megagametogenesis. Moreover, pri-miR398c is transcribed in the female gametophyte but is then translocated to and processed in the ovule sporophytic tissues. Together, SWR1 and ERECTA also activate ARGONAUTE10 (AGO10) expression in the chalaza; AGO10 sequesters miR398, thereby ensuring the expression of three AGAMOUS-LIKE (AGL) genes (AGL51, AGL52, and AGL78) in the female gametophyte. In the context of sexual organ morphogenesis, these findings suggest that the spatiotemporal control of miRNA biogenesis, resulting from coordination between chromatin remodeling and cell signaling, is essential for proper ovule development in Arabidopsis.

Bridging the GAPs in plant reproduction: a comparison of plant and animal GPI-anchored proteins.

KEY MESSAGE: Glycosylphosphatidylinositol (GPI)-anchored proteins (GAPs) are a unique type of membrane-associated proteins in eukaryotes. GPI and GAP biogenesis and function have been well studied in non-plant models and play an important role in the fertility of mouse sperm and egg. Although GPI and GAP biogenesis and function in plants are less known, they are critical for flowering plant reproduction because of their essential roles in the fertility of the male and female gametophytes. In Eukaryotes, GPI, a glycolipid molecule, can be post-translationally attached to proteins to serve as an anchor in the plasma membrane. GPI-anchoring, compared to other modes of membrane attachment and lipidation processes, localizes proteins to the extracellular portion of the plasma membrane and confers several unique attributes including specialized sorting during secretion, molecular painting onto membranes, and enzyme-mediated release of protein through anchor cleavage. While the biosynthesis, structure, and role of GPI are mostly studied in mammals, yeast and protists, the function of GPI and GAPs in plants is being discovered, particularly in gametophyte development and function. Here, we review GPI biosynthesis, protein attachment, and remodeling in plants with insights about this process in mammals. Additionally, we summarize the reproductive phenotypes of all loss of function mutations in Arabidopsis GPI biosynthesis and GAP genes and compare these to the reproductive phenotypes seen in mice to serve as a framework to identify gaps in our understanding of plant GPI and GAPs. In addition, we present an analysis on the gametophyte expression of all Arabidopsis GAPs to assist in further research on the role of GPI and GAPs in all aspects of the gametophyte generation in the life cycle of a plant.

AtPIG-S, a predicted Glycosylphosphatidylinositol Transamidase subunit, is critical for pollen tube growth in Arabidopsis.

BACKGROUND: Glycosylphosphatidylinositol (GPI) addition is one of the several post-translational modifications to proteins that increase their affinity for membranes. In eukaryotes, the GPI transamidase complex (GPI-T) catalyzes the attachment of pre-assembled GPI anchors to GPI-anchored proteins (GAPs) through a transamidation reaction. A mutation in AtGPI8 (gpi8-2), the putative catalytic subunit of GPI-T in Arabidopsis, is transmitted normally through the female gametophyte (FG), indicating the FG tolerates loss of GPI transamidation. In contrast, gpi8-2 almost completely abolishes male gametophyte (MG) function. Still, the unexpected finding that gpi8-2 FGs function normally requires further investigation. Additionally, specific developmental defects in the MG caused by loss of GPI transamidation remain poorly characterized. RESULTS: Here we investigated the effect of loss of AtPIG-S, another GPI-T subunit, in both gametophytes. Like gpi8-2, we showed that a mutation in AtPIG-S (pigs-1) disrupted synergid localization of LORELEI (LRE), a putative GAP critical for pollen tube reception by the FG. Still, pigs-1 is transmitted normally through the FG. Conversely, pigs-1 severely impaired male gametophyte (MG) function during pollen tube emergence and growth in the pistil. A pPIGS:GFP-PIGS transgene complemented these MG defects and enabled generation of pigs-1/pigs-1 seedlings. However, the pPIGS:GFP-PIGS transgene seemingly failed to rescue the function of AtPIG-S in the sporophyte, as pigs-1/pigs-1, pPIGS:GFP-PIGS seedlings died soon after germination. CONCLUSIONS: Characterization of pigs-1 provided further evidence that the FG tolerates loss of GPI transamidation more than the MG and that the MG compared to the FG may be a better haploid system to study the role of GPI-anchoring. Pigs-1 pollen develops normally and thus represent a tool in which GPI anchor biosynthesis and transamidation of GAPs have been uncoupled, offering a potential way to study free GPI in plant development. While previously reported male fertility defects of GPI biosynthesis mutants could have been due either to loss of GPI or GAPs lacking the GPI anchor, our results clarified that the loss of mature GAPs underlie male fertility defects of GPI-deficient pollen grains, as pigs-1 is defective only in the downstream transamidation step.

Workflow to Characterize Mutants with Reproductive Defects.

Reverse genetics approaches for characterizing phenotypes of mutants in a gene of interest (GOI) require thorough genotyping and phenotypic analysis. However, special challenges are encountered when a GOI is expressed in reproductive tissues: a variety of assays are required to characterize the phenotype and a mutant may show sporophytic and/or gametophytic defects in male and/or female reproductive tissues, which are structurally and functionally intertwined. Here, we present a streamlined workflow to characterize mutants with reproductive defects, primarily using Arabidopsis as a model, which can also be adapted to characterize mutants in other flowering plants. Procedures described here can be used to distinguish different kinds of reproductive defects and pinpoint the defective reproductive step(s) in a mutant. Although our procedures emphasize the characterization of mutants with male reproductive defects, they can nevertheless be used to identify female reproductive defects, as those defects could manifest alongside, and sometimes require, male reproductive tissues.

A Fruitful Journey: Pollen Tube Navigation from Germination to Fertilization.

In flowering plants, pollen tubes undergo tip growth to deliver two nonmotile sperm to the ovule where they fuse with an egg and central cell to achieve double fertilization. This extended journey involves rapid growth and changes in gene activity that manage compatible interactions with at least seven different cell types. Nearly half of the genome is expressed in haploid pollen, which facilitates genetic analysis, even of essential genes. These unique attributes make pollen an ideal system with which to study plant cell-cell interactions, tip growth, cell migration, the modulation of cell wall integrity, and gene expression networks. We highlight the signaling systems required for pollen tube navigation and the potential roles of Ca2+ signals. The dynamics of pollen development make sexual reproduction highly sensitive to heat stress. Understanding this vulnerability may generate strategies to improve seed crop yields that are under threat from climate change.

Cellular distribution of secretory pathway markers in the haploid synergid cells of Arabidopsis thaliana.

In flowering plants, cell-cell communication plays a key role in reproductive success, as both pollination and fertilization require pathways that regulate interactions between many different cell types. Some of the most critical of these interactions are those between the pollen tube (PT) and the embryo sac, which ensure the delivery of sperm cells required for double fertilization. Synergid cells function to attract the PT through secretion of small peptides and in PT reception via membrane-bound proteins associated with the endomembrane system and the cell surface. While many synergid-expressed components regulating PT attraction and reception have been identified, few tools exist to study the localization of membrane-bound proteins and the components of the endomembrane system in this cell type. In this study, we describe the localization and distribution of seven fluorescent markers that labelled components of the secretory pathway in synergid cells of Arabidopsis thaliana. These markers were used in co-localization experiments to investigate the subcellular distribution of the two PT reception components LORELEI, a GPI-anchored surface protein, and NORTIA, a MILDEW RESISTANCE LOCUS O protein, both found within the endomembrane system of the synergid cell. These secretory markers are useful tools for both reproductive and cell biologists, enabling the analysis of membrane-associated trafficking within a haploid cell actively involved in polar transport.

KLU suppresses megasporocyte cell fate through SWR1-mediated activation of WRKY28 expression in Arabidopsis.

Germ-line specification is essential for sexual reproduction. In the ovules of most flowering plants, only a single hypodermal cell enlarges and differentiates into a megaspore mother cell (MMC), the founder cell of the female germ-line lineage. The molecular mechanisms restricting MMC specification to a single cell remain elusive. We show that the Arabidopsis transcription factor WRKY28 is exclusively expressed in hypodermal somatic cells surrounding the MMC and is required to repress these cells from acquiring MMC-like cell identity. In this process, the SWR1 chromatin remodeling complex mediates the incorporation of the histone variant H2A.Z at the WRKY28 locus. Moreover, the cytochrome P450 gene KLU, expressed in inner integument primordia, non-cell-autonomously promotes WRKY28 expression through H2A.Z deposition at WRKY28. Taken together, our findings show how somatic cells in ovule primordia cooperatively use chromatin remodeling to restrict germ-line cell specification to a single cell.

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.

Correction: Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil.

[This corrects the article DOI: 10.1371/journal.pgen.1000621.].

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.

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.