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

An Investigation of the Cellular Responses and the Role of the Exocyst Complex in Early Stages of Pollen-Pistil Interactions in Brassicaceae

In Brassicaceae, complex signaling events occur at the early stages of interaction between the pollen grain and the stigmatic surface at the top of the pistil. The initial events of pollen-pistil interactions mediate the recognition of the pollen grain that will lead to the acceptance of a compatible pollen or the rejection of an incompatible pollen. There has been much research into this field in the Brassicaceae, as this family includes many agriculturally important crops; e.g. canola, radish, turnip, cabbage, and many others. My thesis project has focused on the cellular and molecular mechanisms underlying pollen-pistil interactions in the experimentally tractable Arabidopsis thaliana (a self-compatible species). I have also investigated the cellular events regulating pollen-pistil interactions in Arabidopsis lyrata (a self-incompatible species) and Brassica napus Westar and W1 cultivars (a compatible and self-incompatible species, respectively). Compatible pollinations are driven by the ability of the pistil to provide the resources for an acceptable pollen grain to hydrate, germinate and fertilize the ovule. It has been proposed that the delivery of stigmatic resources to the compatible pollen grain is facilitated by polarized vesicle secretion at the pollen-pistil interface. The self-incompatibility response, however, is hypothesized to inhibit the trafficking of stigma-derived compatibility factors to the self pollen to prevent pollen tube entry and hinder fertilization, encouraging genetic diversity within the species. In this thesis, I present my findings on the cytological responses of the stigmatic papillae to the compatible and self-incompatible pollen grains in a detailed time-course. My ultrastructural observations revealed the presence of targeted and localized vesicle secretion at the pollen contact site whereas the absence of secretion after self-incompatible pollination. The octameric exocyst complex is proposed to mediate the aforementioned vesicular trafficking in the stigmatic papillae after compatible pollination. Previously, Exo70A1, a member of the exocyst complex has been identified as an essential component for the acceptance of compatible pollen in A. thaliana. We analyzed the Stigmatic female fertility in RNAi silencing lines for the remaining seven exocyst subunits. Interestingly our novel results uncovered that all eight exocyst subunits are required for the acceptance of compatible pollen in A. thaliana.Ph.D.2017-02-12 00:00:0

Secretory Activity Is Rapidly Induced in Stigmatic Papillae by Compatible Pollen, but Inhibited for Self-Incompatible Pollen in the Brassicaceae

<div><p>[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 <i>Arabidopsis thaliana</i> and <i>Arabidopsis lyrata</i>, vesicle secretion was observed at the stigmatic papillar plasma membrane under the pollen grain while <i>Brassica napus</i> 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 <i>A. thaliana</i> and <i>B. napus</i> 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 <i>A. lyrata</i> and <i>B. napus</i>; 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.</p></div

TEM images of clathrin-coated vesicles in the <i>A. thaliana</i> root tip cells.

<p>(<b>A-C</b>) The root tips from 6-day-old <i>A. thaliana</i> seedlings were observed as a reference for clathrin-coated vesicles (CCV) in the plant endocytic pathway. Clathrin-coated vesicles were observed adjacent to the plasma membrane in the root tip cells in 25/25 samples. Scale bars (A-C) 100 nm.</p

TEM images of <i>B. napus</i> W1 stigmatic papillae in response to self-incompatible pollen.

<p>(<b>A, B</b>) Unpollinated <i>B. napus</i> W1 stigmatic papilla. Secretory activity was not observed at the papillar plasma membrane (PM), and the vacuole was largely clear (no MVBs were visible) in 10/10 samples. (<b>C, D</b>) <i>B. napus</i> W1 stigmatic papilla at 10 min post-pollination with self-incompatible pollen. Secretory activity was not observed at the papillar plasma membrane (PM). Instead, MVBs were observed in the vacuole in 7/10 samples. For 3/10 samples, MVBs were observed in the cytoplasm near the vacuole (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084286#pone.0084286.s002" target="_blank">Figure S2E</a>, F). (<b>E, F</b>) Transgenic <i>B. napus RFP:Exo70A1</i> W1 stigmatic papilla at 10 min post-pollination with self-incompatible pollen. These plants were previously found to have a partial breakdown of the self-incompatibility response due to <i>RFP:Exo70A1</i> expression <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084286#pone.0084286-Samuel1" target="_blank">[17]</a>. Consistent with this partial phenotype, MVBs were observed to be fusing to the plasma membrane in 8/10 samples as shown in (F), and MVBs were also observed in the vacuole in 2/10 samples (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084286#pone.0084286.s002" target="_blank">Figure S2G</a>, H). The white boxed areas in (A, C, E) are shown in the (B, D, F), respectively. Scale bars (A, C, E) 1.5 µm; (B, D, F) 500 nm.</p

Autophagosomes in <i>A. lyrata</i> stigmatic papillae in response to self-incompatible pollen.

<p>(<b>A, B</b>) Florescence microscopy images of MDC stained <i>A. lyrata</i> stigmatic papillae at 10 min post-pollination. Fluorescent signals that may represent autophagosomes were seen in the <i>A. lyrata</i> stigmatic papillae following a self-incompatible pollination (A) in 10/10 samples, but not observed after a cross-compatible pollination (B) in 10/10 samples. (<b>C-F</b>) Confocal microscopy images of transgenic <i>A. lyrata</i> GFP:ATG8a stigmatic papillae at 10 min post-pollination. GFP:ATG8a is a marker for autophagy induction, and GFP signals marking potential autophagosomes were observed in the stigmatic papillae following a self-incompatible pollination (C) in 10/10 samples (corresponding DIC image is shown in D). Punctate GFP signals were not detected within the stigmatic papillae following a cross-compatible pollination (E) in 10/10 samples (corresponding DIC image is shown in F). All samples, including wild-type untransformed <i>A. lyrata</i> stigmatic papillae showed background fluorescence from the cell wall. A  =  autophagosomes; P =  pollen. Scale bars (A, B) 50 µm; (C-F) 10 µm.</p

TEM images of <i>A. thaliana exo70A1-1</i> and <i>B. napus</i> Westar <i>Exo70A1</i> RNAi stigmatic papillae in response to wild-type compatible pollen.

<p><b>(A, B)</b> Unpollinated stigmatic papilla from the <i>A. thaliana exo70A1-1</i> mutant. Some vesicle (V) accumulation was observed in the cytoplasm in 10/10 samples. <b>(C, D)</b> Stigmatic papilla from the <i>A. thaliana exo70A1-1</i> mutant at 10 min following pollination with compatible <i>A. thaliana</i> Col-0 pollen. An accumulation of secretory vesicles (V) in the papillar cytoplasm was observed under the pollen contact site in 10/10 samples. <b>(E, F)</b> Unpollinated stigmatic papilla from the <i>B. napus</i> Westar <i>Exo70A1</i> RNAi R2 line. Some accumulation of MVBs was observed in the cytoplasm in 10/10 samples. <b>(G, H)</b> Stigmatic papilla from the <i>B. napus</i> Westar <i>Exo70A1</i> RNAi R2 line at 10 min following pollination with compatible <i>B. napus</i> Westar pollen. Some MVBs were observed in the papillar cytoplasm in 8/10 samples. For 2/10 samples, MVBs were observed in the cytoplasm and fusing to the plasma membrane (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084286#pone.0084286.s002" target="_blank">Figure S2C</a>, D) which is consistent with these plants displaying an incomplete knockout phenotype <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084286#pone.0084286-Samuel1" target="_blank">[17]</a>. The white boxed areas in (A, C, E, G) are shown in the (B, D, F, H), respectively. Scale bars (A, C, E, G) 1.5 µm; (B, D, F, H) 500 nm.</p

TEM images of <i>A. lyrata</i> stigmatic papillae in response to cross-compatible pollen.

<p>(<b>A, B</b>) Unpollinated stigmatic papilla. Secretory activity was not observed at the papillar plasma membrane (PM) in 10/10 samples. (<b>C, D</b>) Stigmatic papilla at 5 min post-pollination. Secretory activity was not observed at the papillar plasma membrane (PM) in 10/10 samples. (<b>E, F</b>) Stigmatic papilla at 10 min post-pollination. Vesicles (V) were observed to be fusing to the plasma membrane (PM) underneath the pollen contact site in 25/25 samples. (<b>G-H</b>) Pollen tube penetration into the stigmatic papilla at 20 min post-pollination. Vesicles (V) were observed at the papillar plasma membrane (PM) beneath the pollen tube tip in 25/25 samples. The white boxed areas in (A, C, E, G) are shown in the (B, D, F, H), respectively. Scale bars (A, C, E, G) 1.5 µm; (B, D, F, H) 500 nm.</p