Cellular Pathways Regulating Responses to Compatible and Self-Incompatible Pollen inBrassicaandArabidopsisStigmas Intersect at Exo70A1, a Putative Component of the Exocyst Complex

Abstract

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.

Signaling Events in Pollen Acceptance or Rejection in the Arabidopsis Species

“…Thus, these Ca 2+ spikes in the stigmatic papilla underneath the pollen-pistil interface support the premise that the stigmatic papilla responds to the compatible pollen and controls these early postpollination stages. More recently, Exo70A1 has been identifi ed as a factor required in the stigma for the early responses of the stigmatic papilla to the compatible pollen (Samuel et al 2009 ). Exo70A1 is a subunit of the exocyst , an evolutionary conserved protein complex in eukaryotes consisting of eight subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 (Hsu et al 1996 ;TerBush et al 1996 ;Hala et al 2008 ).…”

“…Exo70A1 was identifi ed through work on the self-incompatibility pathway (described in the next section), and this led to the establishment of its role in compatible pollen response in both B. napus and A. thaliana (Samuel et al 2009 ). The A. thaliana exo70A1 mutant displayed a loss of pollen hydration and pollen tube growth when wild-type pollen was placed on the exo70A1 mutant stigma (Samuel et al 2009 ).…”

“…The A. thaliana exo70A1 mutant displayed a loss of pollen hydration and pollen tube growth when wild-type pollen was placed on the exo70A1 mutant stigma (Samuel et al 2009 ). Furthermore, this stigmatic defect was rescued by the stigma-specifi c expression of an RFP:Exo70A1 fusion protein in the A. thaliana exo70A1 mutant.…”

“…Furthermore, this stigmatic defect was rescued by the stigma-specifi c expression of an RFP:Exo70A1 fusion protein in the A. thaliana exo70A1 mutant. Confocal microscopy revealed that RFP:Exo70A1 protein was localized to the apical plasma membrane of mature stigmatic papillae (Samuel et al 2009 ).…”

“…Although RFP:Exo70A1 was localized to the entire stigmatic papillar apical plasma membrane, one would predict that exocyst assembly and vesicle docking would occur in a more localized area, just under the pollen contact site (Samuel et al 2009 ). Consistent with this, we have observed, with compatible pollinations in A. thaliana and A. lyrata , that vesicle-like structures were observed fusing to the papillar plasma membrane under the pollen contact site (Safavian and Goring 2013 ).…”