A new "lock-and-key" system revealed for plant reproductive barriers.

Mate selection in flowering plants can occur very rapidly after male pollen contact on the female pistil, but the cellular regulators driving this process were poorly understood. In this issue of Cell, Lan et al. have discovered the components of a complex ligand-receptor system controlling pollen selection in Arabidopsis thaliana.

Contrasting self-recognition rejection systems for self-incompatibility in Brassica and Papaver.

Self-incompatibility (SI) plays a pivotal role in whether self-pollen is accepted or rejected. Most SI systems employ two tightly linked loci encoding highly polymorphic pollen (male) and pistil (female) S-determinants that control whether self-pollination is successful or not. In recent years our knowledge of the signalling networks and cellular mechanisms involved has improved considerably, providing an important contribution to our understanding of the diverse mechanisms used by plant cells to recognise each other and elicit responses. Here, we compare and contrast two important SI systems employed in the Brassicaceae and Papaveraceae. Both use 'self-recognition' systems, but their genetic control and S-determinants are quite different. We describe the current knowledge about the receptors and ligands, and the downstream signals and responses utilized to prevent self-seed set. What emerges is a common theme involving the initiation of destructive pathways that block the key processes that are required for compatible pollen-pistil interactions.

Autophagy is required for self-incompatible pollen rejection in two transgenic Arabidopsis thaliana accessions.

Successful reproduction in the Brassicaceae is mediated by a complex series of interactions between the pollen and the pistil, and some species have an additional layer of regulation with the self-incompatibility trait. While the initial activation of the self-incompatibility pathway by the pollen S-locus protein 11/S locus cysteine-rich protein and the stigma S Receptor Kinase is well characterized, the downstream mechanisms causing self-pollen rejection are still not fully understood. In previous studies, we detected the presence of autophagic bodies with self-incompatible (SI) pollinations in Arabidopsis lyrata and transgenic Arabidopsis thaliana lines, but whether autophagy was essential for self-pollen rejection was unknown. Here, we investigated the requirement of autophagy in this response by crossing mutations in the essential AUTOPHAGY7 (ATG7) and ATG5 genes into two different transgenic SI A. thaliana lines in the Col-0 and C24 accessions. By using these previously characterized transgenic lines that express A. lyrata and Arabidopsis halleri self-incompatibility genes, we demonstrated that disrupting autophagy weakened their SI responses in the stigma. When the atg7 or atg5 mutations were present, an increased number of SI pollen was found to hydrate and form pollen tubes that successfully fertilized the SI pistils. Additionally, we confirmed the presence of GFP-ATG8a-labeled autophagosomes in the stigmatic papillae following SI pollinations. Together, these findings support the requirement of autophagy in the self-incompatibility response and add to the growing understanding of the intracellular mechanisms employed in the transgenic A. thaliana stigmas to reject self-pollen.

Cell-cell signaling during the Brassicaceae self-incompatibility response.

Self-incompatibility (SI) is a mechanism that many plant families employ to prevent self-fertilization. In the Brassicaceae, the S-haplotype-specific interaction of the pollen-borne ligand, and a stigma-specific receptor protein kinase triggers a signaling cascade that culminates in the rejection of self-pollen. While the upstream molecular components at the receptor level of the signaling pathway have been extensively studied, the intracellular responses beyond receptor activation were not as well understood. Recent research has uncovered several key molecules and signaling events that operate in concert for the manifestation of the self-incompatible responses in Brassicaceae stigmas. Here, we review the recent discoveries in both the compatible and self-incompatible pathways and provide new perspectives on the early stages of Brassicaceae pollen-pistil interactions.

Two subgroups of receptor-like kinases promote early compatible pollen responses in the Arabidopsis thaliana pistil.

In flowering plants, cell-cell communication between the compatible pollen grain/growing pollen tube and the pistil is an essential component for successful sexual reproduction. In Arabidopsis thaliana, the later stages of this dialogue are mediated by several peptide ligands and receptors that guide pollen tubes to the ovules for the release of sperm cells. Despite a detailed understanding of these processes, a key gap remains regarding the nature of the regulators that function at the earlier stages which are essential steps leading to fertilization. Here, we report on new functions for A. thaliana Receptor-Like Kinase (RLK) genes belonging to the LRR-II and LRR-VIII-2 RLK subgroups in the female reproductive tract to regulate compatible pollen hydration and the early stages of pollen tube growth. Mutant pistils for the A. thaliana RKF1 gene cluster were observed to support reduced wild-type pollen hydration and, when combined with the SERK1 and SERK3/BAK1 mutations, reduced pollen tube travel distances occurred. As these mutant pistils displayed a wild-type morphology, we propose that the observed altered compatible pollen responses result from an impaired pollen-pistil dialogue at these early stages.

A Toolkit for Teasing Apart the Early Stages of Pollen-Stigma Interactions in Arabidopsis thaliana.

In hermaphroditic flowering plants, the female pistil serves as the main gatekeeper of mate acceptance as several mechanisms are present to prevent fertilization by unsuitable pollen. The characteristic Brassicaceae dry stigma at the top of pistil represents the first layer that requires pollen recognition to elicit appropriate physiological responses from the pistil. Successful pollen-stigma interactions then lead to pollen hydration, pollen germination, and pollen tube entry into the stigmatic surface. To assess these early stages in detail, our lab has used three experimental procedures to quantitatively and qualitatively characterize the outcome of compatible pollen-stigma interactions that would ultimately lead to the successful fertilization. These assays are also useful for assessing self-incompatible pollinations and mutations that affect these pathways. The model organism, Arabidopsis thaliana, offers an excellent platform for these investigations as loss-of-function or gain-of-function mutants can be easily generated using CRISPR/Cas9 technology, existing T-DNA insertion mutant collections, and heterologous expression constructs, respectively. Here, we provide a detailed description of the methods for these inexpensive assays that can be reliably used to assess pollen-stigma interactions and used to identify new players regulating these processes.

Investigations into a putative role for the novel BRASSIKIN pseudokinases in compatible pollen-stigma interactions in Arabidopsis thaliana.

BACKGROUND: In the Brassicaceae, the early stages of compatible pollen-stigma interactions are tightly controlled with early checkpoints regulating pollen adhesion, hydration and germination, and pollen tube entry into the stigmatic surface. However, the early signalling events in the stigma which trigger these compatible interactions remain unknown. RESULTS: A set of stigma-expressed pseudokinase genes, termed BRASSIKINs (BKNs), were identified and found to be present in only core Brassicaceae genomes. In Arabidopsis thaliana Col-0, BKN1 displayed stigma-specific expression while the BKN2 gene was expressed in other tissues as well. CRISPR deletion mutations were generated for the two tandemly linked BKNs, and very mild hydration defects were observed for wild-type Col-0 pollen when placed on the bkn1/2 mutant stigmas. In further analyses, the predominant transcript for the stigma-specific BKN1 was found to have a premature stop codon in the Col-0 ecotype, but a survey of the 1001 Arabidopsis genomes uncovered three ecotypes that encoded a full-length BKN1 protein. Furthermore, phylogenetic analyses identified intact BKN1 orthologues in the closely related outcrossing Arabidopsis species, A. lyrata and A. halleri. Finally, the BKN pseudokinases were found to be plasma-membrane localized through the dual lipid modification of myristoylation and palmitoylation, and this localization would be consistent with a role in signaling complexes. CONCLUSION: In this study, we have characterized the novel Brassicaceae-specific family of BKN pseudokinase genes, and examined the function of BKN1 and BKN2 in the context of pollen-stigma interactions in A. thaliana Col-0. Additionally, premature stop codons were identified in the predicted stigma specific BKN1 gene in a number of the 1001 A. thaliana ecotype genomes, and this was in contrast to the out-crossing Arabidopsis species which carried intact copies of BKN1. Thus, understanding the function of BKN1 in other Brassicaceae species will be a key direction for future studies.

Identification of a role for an E6-like 1 gene in early pollen-stigma interactions in Arabidopsis thaliana.

KEY MESSAGE: We describe a function for a novel Arabidopsis gene, E6-like 1 (E6L1), that was identified as a highly expressed gene in the stigma and plays a role in early post-pollination stages. In Arabidopsis, successful pollen-stigma interactions are dependent on rapid recognition of compatible pollen by the stigmatic papillae located on the surface of the pistil and the subsequent regulation of pollen hydration and germination, and followed by the growth of pollen tubes through the stigma surface. Here we have described the function of a novel gene, E6-like 1 (E6L1), that was identified through the analysis of transcriptome datasets, as one of highest expressed genes in the stigma, and furthermore, its expression was largely restricted to the stigma and trichomes. The first E6 gene was initially identified as a highly expressed gene during cotton fiber development, and related E6-like predicted proteins are found throughout the Angiosperms. To date, no orthologous genes have been assigned a biological function. Both the Arabidopsis E6L1 and cotton E6 proteins are predicted to be secreted, and this was confirmed using an E6L1:RFP fusion construct. To further investigate E6L1's function, one T-DNA and two independent CRISPR-generated mutants were analyzed for compatible pollen-stigma interactions, and pollen hydration, pollen adhesion, and seed set were mildly impaired for the e6l1 mutants. This work identifies E6L1 as a novel stigmatic factor that plays a role during the early post-pollination stages in Arabidopsis.

The Molecular and Cellular Regulation of Brassicaceae Self-Incompatibility and Self-Pollen Rejection.

In flowering plants, sexual reproduction is actively regulated by cell-cell communication between the male pollen and female pistil, and many species possess self-incompatibility systems for the selective rejection of self-pollen to maintain genetic diversity. The Brassicaceae self-incompatibility pathway acts early on when pollen grains have landed on the stigmatic papillae at the top of the pistil. Extensive studies have revealed that self-pollen rejection in the Brassicaceae is initiated by an S-haplotype-specific interaction between two polymorphic proteins: the pollen S-locus protein 11/S cysteine-rich (SP11/SCR) ligand and the stigma S receptor kinase (SRK). While the different S-haplotypes are typically codominant, there are several examples of dominant-recessive interactions, and a small RNA-based regulation of SP11/SCR expression has been uncovered as a mechanism behind these genetic interactions. Recent research has also added to our understanding of various cellular components in the pathway leading from the SP11/SCR-SRK interaction, including two signaling proteins, the M-locus protein kinase (MLPK) and the ARM-repeat containing 1 (ARC1) E3 ligase, as well as calcium fluxes and induction of autophagy in the stigmatic papillae. Finally, a better understanding of the compatible pollen responses that are targeted by the self-incompatibility pathway is starting to emerge, and this will allow us to more fully understand how the Brassicaceae self-incompatibility pathway causes self-pollen rejection. Here, we provide an overview of the field, highlighting recent contributions to our understanding of Brassicaceae self-incompatibility, and draw comparisons to a recently discovered unilateral incompatibility system.

Pollen Acceptance or Rejection: A Tale of Two Pathways.

While the molecular and cellular basis of self-incompatibility leading to self-pollen rejection in the Brassicaceae has been extensively studied, relatively little attention has been paid to compatible pollen recognition and the corresponding cellular responses in the stigmatic papillae. This is now changing because research has started to uncover steps in the Brassicaceae 'basal compatible pollen response pathway' in the stigma leading to pollen hydration and germination. Furthermore, recent studies suggest that self-incompatible pollen activates both the basal compatible pathway and the self-incompatibility pathway in the stigma, with the self-incompatibility response ultimately prevailing to reject self-pollen. We review here recent discoveries in both pathways and discuss how compatible pollen is accepted by the stigma versus the rejection of self-incompatible pollen.

Following the Time-Course of Post-pollination Events by Transmission Electron Microscopy (TEM): Buildup of Exosome-Like Structures with Compatible Pollinations.

In the Brassicaceae, the dry stigma is an initial barrier to pollen acceptance as the stigmatic papillae lack surface secretions, and consequently rapid cellular responses are required to accept compatible pollen. Regulated secretion with secretory vesicles or multivesicular bodies is initiated in the stigmatic papillae towards the compatible pollen grain. In self-incompatible species, this basal compatible pollen response is superseded by the self-incompatibility signaling pathway where the secretory organelles are found in autophagosomes and vacuole for destruction. In this chapter, we describe a detailed protocol using the Transmission Electron Microscope to document the rapid cellular changes that occur in the stigmatic papillae in response to compatible versus self-incompatible pollen, at the pollen-stigma interface.

RNA Silencing of Exocyst Genes in the Stigma Impairs the Acceptance of Compatible Pollen in Arabidopsis.

Initial pollen-pistil interactions in the Brassicaceae are regulated by rapid communication between pollen grains and stigmatic papillae and are fundamentally important, as they are the first step toward successful fertilization. The goal of this study was to examine the requirement of exocyst subunits, which function in docking secretory vesicles to sites of polarized secretion, in the context of pollen-pistil interactions. One of the exocyst subunit genes, EXO70A1, was previously identified as an essential factor in the stigma for the acceptance of compatible pollen in Arabidopsis (Arabidopsis thaliana) and Brassica napus. We hypothesized that EXO70A1, along with other exocyst subunits, functions in the Brassicaceae dry stigma to deliver cargo-bearing secretory vesicles to the stigmatic papillar plasma membrane, under the pollen attachment site, for pollen hydration and pollen tube entry. Here, we investigated the functions of exocyst complex genes encoding the remaining seven subunits, SECRETORY3 (SEC3), SEC5, SEC6, SEC8, SEC10, SEC15, and EXO84, in Arabidopsis stigmas following compatible pollinations. Stigma-specific RNA-silencing constructs were used to suppress the expression of each exocyst subunit individually. The early postpollination stages of pollen grain adhesion, pollen hydration, pollen tube penetration, seed set, and overall fertility were analyzed in the transgenic lines to evaluate the requirement of each exocyst subunit. Our findings provide comprehensive evidence that all eight exocyst subunits are necessary in the stigma for the acceptance of compatible pollen. Thus, this work implicates a fully functional exocyst complex as a component of the compatible pollen response pathway to promote pollen acceptance.

Secretory activity is rapidly induced in stigmatic papillae by compatible pollen, but inhibited for self-incompatible pollen in the Brassicaceae.

[In the Brassicaceae, targeted exocytosis to the stigmatic papillar plasma membrane under the compatible pollen grain is hypothesized to be essential for pollen hydration and pollen tube penetration. In contrast, polarized secretion is proposed to be inhibited in the stigmatic papillae during the rejection of self-incompatible pollen. Using transmission electron microscopy (TEM), we performed a detailed time-course of post-pollination events to view the cytological responses of the stigmatic papillae to compatible and self-incompatible pollinations. For compatible pollinations in Arabidopsis thaliana and Arabidopsis lyrata, vesicle secretion was observed at the stigmatic papillar plasma membrane under the pollen grain while Brassica napus stigmatic papillae appeared to use multivesicular bodies (MVBs) for secretion. Exo70A1, a component of the exocyst complex, has been previously implicated in the compatible pollen responses, and disruption of Exo70A1 in both A. thaliana and B. napus resulted in a loss of secretory vesicles/MVBs at the stigmatic papillar plasma membrane. Similarly, for self-incompatible pollinations, secretory vesicles/MVBs were absent from the stigmatic papillar plasma membrane in A. lyrata and B. napus; and furthermore, autophagy appeared to be induced to direct vesicles/MVBs to the vacuole for degradation. Thus, these findings support a model where the basal pollen recognition pathway in the stigmatic papilla promotes exocytosis to accept compatible pollen, and the basal pollen recognition pathway is overridden by the self-incompatibility pathway to prevent exocytosis and reject self-pollen.

The ARC1 E3 ligase gene is frequently deleted in self-compatible Brassicaceae species and has a conserved role in Arabidopsis lyrata self-pollen rejection.

Self-pollen rejection is an important reproductive regulator in flowering plants, and several different intercellular signaling systems have evolved to elicit this response. In the Brassicaceae, the self-incompatibility system is mediated by the pollen S-locus Cys-Rich/S-locus Protein11 (SCR/SP11) ligand and the pistil S Receptor Kinase (SRK). While the SCR/SP11-SRK recognition system has been identified in several species across the Brassicaceae, less is known about the conservation of the SRK-activated cellular responses in the stigma, following self-pollen contact. The ARM Repeat Containing1 (ARC1) E3 ubiquitin ligase functions downstream of SRK for the self-incompatibility response in Brassica, but it has been suggested that ARC1 is not required in Arabidopsis species. Here, we surveyed the presence of ARC1 orthologs in several recently sequenced genomes from Brassicaceae species that had diversified ∼20 to 40 million years ago. Surprisingly, the ARC1 gene was deleted in several species that had lost the self-incompatibility trait, suggesting that ARC1 may lose functionality in the transition to self-mating. To test the requirement of ARC1 in a self-incompatible Arabidopsis species, transgenic ARC1 RNA interference Arabidopsis lyrata plants were generated, and they exhibited reduced self-incompatibility responses resulting in successful fertilization. Thus, this study demonstrates a conserved role for ARC1 in the self-pollen rejection response within the Brassicaceae.

Proteomic analysis of Brassica stigmatic proteins following the self-incompatibility reaction reveals a role for microtubule dynamics during pollen responses.

Mate selection and maintenance of genetic diversity is crucial to successful reproduction and species survival. Plants utilize self-incompatibility system as a genetic barrier to prevent self pollen from developing on the pistil, leading to hybrid vigor and diversity. In Brassica (canola, kale, and broccoli), an allele-specific interaction between the pollen SCR/SP11 (S-locus cysteine rich protein/S locus protein 11) and the pistil S Receptor Kinase, results in the activation of SRK which recruits the Arm Repeat Containing 1 (ARC1) E3 ligase to the proteasome. The targets of Arm Repeat Containing 1 are proposed to be compatibility factors, which when targeted for degradation by Arm Repeat Containing 1 results in pollen rejection. Despite the fact that protein degradation is predicted to be important for successful self-pollen rejection, the identity of the various proteins whose abundance is altered by the SI pathway has remained unknown. To identify potential candidate proteins regulated by the SI response, we have used the two-dimensional difference gel electrophoresis analysis, coupled with matrix-assisted laser desorption ionization/time of flight/MS. We identified 56 differential protein spots with 19 unique candidate proteins whose abundance is down-regulated following self-incompatible pollinations. The identified differentials are predicted to function in various pathways including biosynthetic pathways, signaling, cytoskeletal organization, and exocytosis. From the 19 unique proteins identified, we investigated the role of tubulin and the microtubule network during both self-incompatible and compatible pollen responses. Moderate changes in the microtubule network were observed with self-incompatible pollinations; however, a more distinct localized break-down of the microtubule network was observed during compatible pollinations, that is likely mediated by EXO70A1, leading to successful pollination.

Misregulation of phosphoinositides in Arabidopsis thaliana decreases pollen hydration and maternal fertility.

Phosphoinositides are important lipids involved in membrane identity, vesicle trafficking, and intracellular signaling. In recent years, phosphoinositides have been shown to play a critical role in polarized secretion in plants, as perturbations of phosphoinositide metabolism, through loss of function mutants, result in defects in root hair elongation and pollen tube growth, where polarized secretion occurs rapidly. In the Brassicaceae, responses of stigmatic papillae to compatible pollen are also thought to involve highly regulated secretory events to facilitate pollen hydration and penetration of the pollen tube through the stigmatic surface. We therefore sought to analyze the female sporophyte fertility of the root hair defective4-1 mutant and the PI 4-kinase ß1/ß2 double mutant, which differentially affect phosphatidylinositol-4-phosphate (PI4P) levels. Stigmas from both mutants supported slower rates of pollen grain hydration, and the fecundity of these mutants was also diminished as a result of failed pollination events. This study therefore concludes that PI4P is integral to appropriate pistil responses to compatible pollen.

Pollen-pistil interactions regulating successful fertilization in the Brassicaceae.

In the Brassicaceae, the acceptance of compatible pollen and the rejection of self-incompatible pollen by the pistil involves complex molecular communication systems between the pollen grain and the female reproductive structures. Preference towards species related-pollen combined with self-recognition systems, function to select the most desirable pollen; and thus, increase the plant's chances for the maximum number of successful fertilizations and vigorous offspring. The Brassicaceae is an ideal group for studying pollen-pistil interactions as this family includes a diverse group of agriculturally relevant crops as well as several excellent model organisms for studying both compatible and self-incompatible pollinations. This review will describe the cellular systems in the pistil that guide the post-pollination events, from pollen capture on the stigmatic papillae to pollen tube guidance to the ovule, with the final release of the sperm cells to effect fertilization. The interplay of other recognition systems, such as the self-incompatibility response and interspecific interactions, on regulating post-pollination events and selecting for compatible pollen-pistil interactions will also be explored.

Cellular pathways regulating responses to compatible and self-incompatible pollen in Brassica and Arabidopsis stigmas intersect at Exo70A1, a putative component of the exocyst complex.

In the Brassicaceae, compatible pollen-pistil interactions result in pollen adhesion to the stigma, while pollen grains from unrelated plant species are largely ignored. There can also be an additional layer of recognition to prevent self-fertilization, the self-incompatibility response, whereby self pollen grains are distinguished from nonself pollen grains and rejected. This pathway is activated in the stigma and involves the ARM repeat-containing 1 (ARC1) protein, an E3 ubiquitin ligase. In a screen for ARC1-interacting proteins, we have identified Brassica napus Exo70A1, a putative component of the exocyst complex that is known to regulate polarized secretion. We show through transgenic studies that loss of Exo70A1 in Brassica and Arabidopsis thaliana stigmas leads to the rejection of compatible pollen at the same stage as the self-incompatibility response. A red fluorescent protein:Exo70A1 fusion rescues this stigmatic defect in Arabidopsis and is found to be mobilized to the plasma membrane concomitant with flowers opening. By contrast, increased expression of Exo70A1 in self-incompatible Brassica partially overcomes the self pollen rejection response. Thus, our data show that the Exo70A1 protein functions at the intersection of two cellular pathways, where it is required in the stigma for the acceptance of compatible pollen in both Brassica and Arabidopsis and is negatively regulated by Brassica self-incompatibility.

Antisense suppression of thioredoxin h mRNA in Brassica napus cv. Westar pistils causes a low level constitutive pollen rejection response.

In Brassica , the thioredoxin h proteins, THL1 and THL2, were previously found to be potential inhibitors of the S receptor kinase (SRK) in the Brassica self-incompatibility response. To investigate the biological roles of THL1 and THL2 in pollen-pistil interactions, the stigma-specific SLR1 promoter was used to drive antisense THL1/2 expression in Brassica napus cv. Westar. This cultivar is normally compatible, but antisense suppression of THL1/2 led to a low level constitutive rejection of all Brassica napus pollen tested. Fluorescence microscopy revealed that the pollen rejection was a typical Brassica self-incompatibility rejection response with reduced pollen adhesion, germination and pollen tube growth. In addition, Westar was found to express the SLG(15) and SRK(15) proteins which may be the target of regulation by THL1 and THL2. Thus, these results indicate that the THL1 and THL2 are required for full pollen acceptance in B. napus cv. Westar.