Showing papers by "Daphne R. Goring published in 2020"

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

Abstract: 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.

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

Abstract: 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 protein11/S-locus cysteine-rich peptide 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 had detected the presence of autophagic bodies with self-incompatible pollinations in Arabidopsis lyrata and transgenic A. thaliana lines, but it was not known if autophagy was essential for self-pollen rejection. Here, we investigated the requirement of autophagy in this response by crossing mutations in the essential AUTOPHAGY7 (ATG7) gene into two different transgenic self-incompatible A. thaliana lines in the Col-0 and C24 accessions. By using these previously characterized transgenic lines that express A. lyrata and A. halleri self-incompatibility genes, we demonstrated that disrupting autophagy can weaken their self-incompatible responses in the stigma. When the atg7 mutation was present, an increased number of self-incompatible pollen were found to hydrate and form pollen tubes that successfully fertilized the self-incompatible pistils. Additionally, we confirmed the presence of GFP-ATG8a labelled autophagosomes in the stigmatic papillae following self-incompatible pollinations. Together, these findings support the requirement of autophagy in the self-incompatibility response and add to the growing understanding of the cellular events that take place in the stigma to reject self-pollen. One Sentence Summary In self-incompatible transgenic Arabidopsis thaliana lines, autophagy is an integral part of the cellular responses in the stigma to efficiently block fertilization by self-incompatible pollen.

Two Groups of Arabidopsis Receptor Kinases Preferentially Regulate the Growth of Intraspecies Pollen Tubes in the Female Reproductive Tract

Abstract: In flowering plants, continuous cell-cell communication between the compatible male pollen grain/growing pollen tube and the female pistil is required for successful sexual reproduction. In Arabidopsis thaliana, the later stages of this dialogue are mediated by several peptide ligands and receptor kinases that guide pollen tubes to the ovules for the release of sperm cells. Despite a detailed understanding of these processes, a key gap remains on the nature of the regulators that function at the earlier stages. Here, we report on two groups of A. thaliana receptor kinases, the LRR-VIII-2 RK subclass and the SERKs, that function in the female reproductive tract to regulate the compatible pollen grains and early pollen tube growth, both essential steps for the downstream processes leading to fertilization. Multiple A. thaliana LRR-VIII-2 RK and SERK knockout mutant combinations were created, and several phenotypes were observed such as reduced wild-type pollen hydration and reduced pollen tube travel distances. As these mutant pistils displayed a wild-type morphology, the observed altered responses of the wild-type pollen are proposed to result from the loss of these receptor kinases leading to an impaired pollen-pistil dialogue at these early stages. Furthermore, using pollen from related Brassicaceae species, we also discovered that these receptor kinases are required in the female reproductive tract to establish a reproductive barrier to interspecies pollen. Thus, we propose that the LRR-VIII-2 RKs and the SERKs play a dual role in the preferential selection and promotion of intraspecies pollen over interspecies pollen.

Two groups of Arabidopsis receptor kinases preferentially regulate the growth of intraspecies pollen tubes in the female reproductive tract

Abstract: Summary In flowering plants, continuous cell-cell communication between the compatible male pollen grain/growing pollen tube and the female pistil is required for successful sexual reproduction. In Arabidopsis thaliana, the later stages of this dialogue are mediated by several peptide ligands and receptor kinases that guide pollen tubes to the ovules for the release of sperm cells. Despite a detailed understanding of these processes, a key gap remains on the nature of the regulators that function at the earlier stages. Here, we report on two groups of A. thaliana receptor kinases, the LRR-VIII-2 RK subclass and the SERKs, that function in the female reproductive tract to regulate the compatible pollen grains and early pollen tube growth, both essential steps for the downstream processes leading to fertilization. Multiple A. thaliana LRR-VIII-2 RK and SERK knockout mutant combinations were created, and several phenotypes were observed such as reduced wild-type pollen hydration and reduced pollen tube travel distances. As these mutant pistils displayed a wild-type morphology, the observed altered responses of the wild-type pollen are proposed to result from the loss of these receptor kinases leading to an impaired pollen-pistil dialogue at these early stages. Furthermore, using pollen from related Brassicaceae species, we also discovered that these receptor kinases are required in the female reproductive tract to establish a reproductive barrier to interspecies pollen. Thus, we propose that the LRR-VIII-2 RKs and the SERKs play a dual role in the preferential selection and promotion of intraspecies pollen over interspecies pollen.