Investigations into a putative role for the novel BRASSIKIN pseudokinases in compatible pollen-stigma interactions in Arabidopsis thaliana.

BACKGROUND: 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. However, the early signalling events in the stigma which trigger these compatible interactions remain unknown. RESULTS: A 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. CONCLUSION: In 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

Altered Germination and Subcellular Localization Patterns for PUB44/SAUL1 in Response to Stress and Phytohormone Treatments

BACKGROUND: In plants, the ubiquitin-proteasome system is emerging as a significant regulatory system throughout the plant lifecycle. The ubiquitination of a target protein requires the sequential actions of the E1, E2 and E3 enzymes, with the latter E3 enzyme conferring target selection in this process. There are a large number of predicted E3 enzymes in plant genomes, and very little is known about the functions of many of these predicted genes. Here we report here an analysis of two closely-related members of the Arabidopsis Plant U-box (PUB) family of E3 ubiquitin ligases, PUB43 and PUB44. PRINCIPAL FINDINGS: Homozygous pub44/pub44 mutant seedlings were found displayed a seedling lethal phenotype and this corresponded with widespread cell death lesions throughout the cotyledons and roots. Interestingly, heterozygous PUB44/pub44 seedlings were wild-type in appearance yet displayed intermediate levels of cell death lesions in comparison to pub44/pub44 seedlings. In contrast, homozygous pub43/pub43 mutants were viable and did not show any signs of cell death despite the PUB43 gene being more highly expressed than PUB44. The PUB44 mutants are not classical lesion mimic mutants as they did not have increased resistance to plant pathogens. We also observed increased germination rates in mutant seeds for both PUB44 and PUB43 under inhibitory concentrations of abscisic acid. Finally, the subcellular localization of PUB44 was investigated with transient expression assays in BY-2 cells. Under varying conditions, PUB44 was observed to be localized to the cytoplasm, plasma membrane, or nucleus. CONCLUSIONS: Based on mutant plant analyses, the Arabidopsis PUB43 and PUB44 genes are proposed to function during seed germination and early seedling growth. Given PUB44's ability to shuttle from the nucleus to the plasma membrane, PUB44 may be active in different subcellular compartments as part of these biological functions

The role of autophagy in the Arabidopsis self-incompatible pollen rejection response

In plants, macroautophagy/autophagy is an essential mechanism responsible for a large variety of processes throughout the plant’s lifecycle, including nutrient processing, immunity, stress responses and senescence. Previous studies had observed the presence of autophagosomes in an Arabidopsis sexual reproduction system that prevents self-fertilization (self-incompatibility), but their requirement in this pathway was unclear. Using autophagy-deficient mutants, we have recently found that autophagy is a key contributor in the Arabidopsis self-incompatibility response to reject self-pollen

Secretory Activity Is Rapidly Induced in Stigmatic Papillae by Compatible Pollen, but Inhibited for Self-Incompatible Pollen in the Brassicaceae

<div><p>[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 <i>Arabidopsis thaliana</i> and <i>Arabidopsis lyrata</i>, vesicle secretion was observed at the stigmatic papillar plasma membrane under the pollen grain while <i>Brassica napus</i> 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 <i>A. thaliana</i> and <i>B. napus</i> 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 <i>A. lyrata</i> and <i>B. napus</i>; 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.</p></div

TEM images of <i>B. napus</i> Westar stigmatic papillae in response to self-compatible pollen.

<p>(<b>A, B</b>) Unpollinated stigmatic papilla. Secretory activity was not observed at the papillar plasma membrane (PM) in 10/10 samples. (<b>C, D</b>) Stigmatic papilla at 5 min post-pollination showing an MVB fusing to the plasma membrane (PM). MVBs at the plasma membrane were observed in 10/10 samples. (<b>E, F</b>) Stigmatic papilla at 10 min post-pollination showing several MVBs fusing to the plasma membrane (PM) underneath the pollen contact site. MVBs at the plasma membrane were observed in 23/25 samples. For 2/25 samples, vesicles were observed to be fusing to the plasma membrane (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084286#pone.0084286.s002" target="_blank">Figure S2A</a>, B). (<b>G, H</b>) Pollen tube penetration into the stigmatic papilla at 20 min post-pollination. The material under the papillar cell wall appears to be the exosomes (E) released from the MVBs. This pattern was observed in 15/15 samples. The white boxed areas in (A, C, E, G) are shown in the (B, D, F, H), respectively. Scale bars (A, C, E, G) 1.5 µm; (B, D, F, H) 500 nm.</p

TEM images of clathrin-coated vesicles in the <i>A. thaliana</i> root tip cells.

<p>(<b>A-C</b>) The root tips from 6-day-old <i>A. thaliana</i> seedlings were observed as a reference for clathrin-coated vesicles (CCV) in the plant endocytic pathway. Clathrin-coated vesicles were observed adjacent to the plasma membrane in the root tip cells in 25/25 samples. Scale bars (A-C) 100 nm.</p