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.

The Arabidopsis SNARE complex genes regulate the early stages of pollen-stigma interactions.

KEY MESSAGE: The VAMP721, VAMP722, SYP121, SYP122 and SNAP33 SNAREs are required in the Arabidopsis stigma for pollen hydration, further supporting a role for vesicle trafficking in the stigma's pollen responses. In the Brassicaceae, the process of accepting compatible pollen is a key step in successful reproduction and highly regulated following interactions between the pollen and the stigma. Central to this is the initiation of secretion in the stigma, which is proposed to provide resources to the pollen for hydration and germination and pollen tube growth. Previously, the eight exocyst subunit genes were shown to be required in the Arabidopsis stigma to support these pollen responses. One of the roles of the exocyst is to tether secretory vesicles at the plasma membrane for membrane fusion by the SNARE complex to enable vesicle cargo release. Here, we investigate the role of Arabidopsis SNARE genes in the stigma for pollen responses. Using a combination of different knockout and knockdown SNARE mutant lines, we show that VAMP721, VAMP722, SYP121, SYP122 and SNAP33 are involved in this process. Significant disruptions in pollen hydration were observed following pollination of wildtype pollen on the mutant SNARE stigmas. Overall, these results place the Arabidopsis SNARE complex as a contributor in the stigma for pollen responses and reaffirm the significance of secretion in the stigma to support the pollen-stigma interactions.