[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.

Flowering plants have evolved various strategies for avoiding self-pollen to drive genetic diversity. These strategies include spatially separated sexual organs (herkogamy), timing differences between male pollen release and female pistil receptivity (dichogamy), and self-pollen rejection. Within the Brassicaceae, these outcrossing systems are the evolutionary default state, and many species display these traits, including Arabidopsis lyrata. In contrast to A. lyrata, closely related Arabidopsis thaliana has lost these self-pollen traits and thus represents an excellent system to test genes for reconstructing these evolutionary traits. We previously demonstrated that the ARC1 E3 ligase is required for self-incompatibility in two diverse Brassicaceae species, Brassica napus and A. lyrata, and is frequently deleted in self-compatible species, including A. thaliana. In this study, we examined ARC1's requirement for reconstituting self-incompatibility in A. thaliana and uncovered an important role for ARC1 in promoting a strong and stable pollen rejection response when expressed with two other A. lyrata self-incompatibility factors. Furthermore, we discovered that ARC1 promoted an approach herkogamous phenotype in A. thaliana flowers. Thus, ARC1's expression resulted in two different A. lyrata traits for self-pollen avoidance and highlights the key role that ARC1 plays in the evolution and retention of outcrossing systems.

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 RNAsilencing 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.

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We investigate the anatomy and fine structure of styles and pollen tubes of two distylous Turnera species, which possess a heteromorphic self-incompatibility system. We use fluorescence and transmission electron microscopy to provide the first description of the cellular aspects of pollen-pistil interactions and ultrastructural changes to pollen tubes during the self-incompatibility response of the morphs. No obvious ultrastructural differences occur between pistils and compatible pollen tubes. Conspicuous differences were, however, observed between incompatible pollen tubes of the morphs. Incompatible pollen tubes of the long-styled morph always appear to be intact, and pollen tube tips are often highly fluorescent when stained with aniline blue, a stain for callose. Swelling and loss of cristae of mitochondria, and circular rough endoplasmic reticulum, were observed for incompatible pollen tubes of the long-styled morph. For incompatible pollen tubes of the short-styled morph, the tube cell wall apex and plasma membrane often appear to be ruptured and no easily recognizable organelles, such as mitochondria, can be discerned. Our results clearly show ultrastructural differences between the morphs and support the hypothesis that different self-incompatibility mechanisms might operate between them.