Abstract
BackgroundIn 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.ResultsA 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.ConclusionIn 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.
Highlights
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
The gene, At5g11400, was a top hit (Additional file 2: Table S1) and displayed stigma-specific expression in the transcriptome datasets (Additional file 1: Figure S1, [35, 43, 44]). This gene is predicted to encode a novel receptor-like cytoplasmic kinase (RLCK) which we named BRASSIKIN 1 (BKN1)
In this study, we have investigated a novel family of pseudokinase genes, the BRASSIKINs (BKNs) that are only found in core-Brassicaceae species
Summary
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. In the Brassicaceae, the early post-pollination stages of pollen adhesion and hydration, and pollen tube entry into the stigma are highly-regulated and represent the first of several stages leading to the release of the sperm cells at the ovule for fertilization (reviewed in [1,2,3,4,5]). The Brassicaceae stigma surface is covered with unicellular stigmatic papillae, and the process of pollen capture is very rapid, occurring in as little as 30 s following a compatible pollination in Arabidopsis thaliana [8]. Perception of peptide ligands by receptor kinases plays a prominent role in the regulation of downstream compatible pollenpistil interactions and pollen tube guidance, as well as the rejection of self-pollen in self-incompatible Brassicaceae species (reviewed in [1, 2, 4, 5, 16])
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