[Abstract unavailable

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.

ARC1 is a novel U-box protein required in the Brassica pistil for the rejection of self-incompatible pollen; it functions downstream of the S receptor kinase (SRK). Here, we show that ARC1 has E3 ubiquitin ligase activity and contains several motifs that influence its subcellular localization. ARC1 can shuttle between the nucleus, cytosol, and proteasome/COP9 signalosome (CSN) when expressed in tobacco BY-2 suspension-cultured cells. However, ARC1 localization to the proteasome/ CSN occurs only in the presence of an active SRK. In the pistil, ubiquitinated protein levels increase specifically with incompatible pollinations, but they do not change in ARC1 antisense-suppressed pistils. In addition, inhibition of the proteasomal proteolytic activity disrupts the self-incompatibility response. We propose that ARC1 promotes the ubiquitination and proteasomal degradation of compatibility factors in the pistil, which in turn leads to pollen rejection.

Screening of a yeast two-hybrid library for proteins that interact with the kinase domain of an S-locus receptor kinase (SRK) resulted in the isolation of a plant protein called ARC1 (Arm Repeat Containing). This interaction was mediated by the C-terminal region of ARC1 in which five arm repeat units were identified. Using the yeast twohybrid system and in vitro binding assays, ARC1 was found to interact specifically with the kinase domains from SRK-910 and SRK-A14 but failed to interact with kinase domains from two different Arabidopsis receptor-like kinases. In addition, treatment with a protein phosphatase or the use of a kinaseinactive mutant reduced or abolished the binding of ARC1 to the SRK-910 kinase domain, indicating that the interaction was phosphorylation dependent. Lastly, RNA blot analysis revealed that the expression of ARC1 is restricted to the stigma, the site of the self-incompatibility response.Many flowering plants employ self-incompatibility systems to prevent inbreeding and promote outcrossing (for review, see refs. 1-3). In the Brassica family, the sporophytic selfincompatibility system is controlled by the multi-allelic S locus (4). When the pollen parent and the pistil share the same S allele, pollen germination or pollen tube growth is inhibited, thereby leading to failure of fertilization. Molecular and biochemical characterization of the S locus has led to the isolation of two genes, the S-locus glycoprotein (SLG) and S-locus receptor kinase (SRK) genes, both of which are highly expressed in the pistil (5, 6). These two genes are tightly linked, and for one S-allele, the SLG and SRK were found within a region of 30 kb (7). Within the pistil, the SLG has been localized to the cell wall of the stigma papillae, whereas the SRK is present as a transmembrane protein in the stigma (8-10). Based on DNA sequence comparisons, the N-terminal structure of the SRK gene resembles the SLG coding region, and its C-terminal portion encodes a putative serine͞threonine kinase (6). Further characterization of the kinase domain has confirmed that the SRK encoded a functional serine͞ threonine kinase (11,12).Both SLG and SRK have been shown to be determinants of self-incompatibility in Brassica, as either loss of SLG gene expression (13-15) or mutations in the SRK gene (16, 17) were found to be associated with self-compatibility. As the S locus determines the specific interaction between the pollen and stigma, it has been postulated that the pollen component of the self-incompatibility system may be encoded by a functional gene linked to the SLG and SRK genes. One candidate for this pollen component is the anther-specific SLA gene (18). The pollen component on the incompatible pollen may serve as a ligand, which is recruited likely by the SLG to interact with the SRK. The specific interaction between the ligand and SRK would in turn activate the SRK and trigger a signaling cascade, leading to pollen rejection by the stigma papillae cells (11,19,20). Little is understood about the molecular mechani...

Ubiquitin-mediated proteolysis is an integral part of diverse cellular functions, and of the three enzymes involved in linking ubiquitin to protein targets, the E3 ubiquitin ligases are of particular interest as they confer substrate specificity during this process. The E3 ubiquitin ligases can be categorized based on mechanism of action and on the presence of specific domains such as RING, HECT, F-box, and U-box. In plants, the U-box family has undergone a large gene expansion that may be attributable to biological processes unique to the plant life cycle. For example, there are 64 predicted plant U-box (PUB) proteins in Arabidopsis, and the biological roles of many of these have yet to be determined. Research on PUB genes from several different plants has started to elucidate a range of functions for this family, from self-incompatibility and hormone responses to defence and abiotic stress responses. Expression profiling has also been used as a starting point to elucidate PUB function, and has uncovered a strong connection of PUB genes to various stress responses. Finally, some PUB proteins have been linked to receptor kinases as upstream activators, and downstream target substrates are also starting to emerge. The mechanisms of action range from the observation of mono-ubiquitination during non-proteolytic signalling to directed regulation of proteasomal components during stress responses, and cell death appears to be a theme underlying many PUB functions.

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.ARM repeats are short 42-amino acid motifs that were first identified in the fruitfly (Drosophila melanogaster) segment polarity protein, armadillo (Riggleman et al., 1989). ARM repeats have been subsequently identified in a wide range of eukaryotic proteins, and these proteins interact with numerous other proteins via their ARM repeats, resulting in the regulation of a variety of cellular processes (for review, see Hatzfeld, 1999). Based on the crystal structure of the mammalian armadillo homolog, ␤-catenin, each ARM repeat forms a trihelical structure that folds into a superhelix, and six ARM repeats are proposed to constitute a protein interaction domain (Huber et al., 1997). In animals, wellcharacterized ARM repeat proteins include ␤-catenin/armadillo involved in the Wnt/wingless signaling pathway and cadherin-mediated cell adhesion, the APC tumor suppressor protein in Wnt signaling, and several other cadherin-associated ARM repeat proteins (Hatzfeld, 1999). In addition, there is the conserved nuclear import pathway in yeast (Saccharomyces cerevisiae), plants, and animals that involves the ARM repeat protein, Importin-␣, and the Importin-␤ protein with related HEAT repeats (Andrade et al., 2001).More recently, a new class of ARM repeat proteins was identified in plants where the ARM repeat region is preceded by a E3 ubiquitin ligase motif called the U-box (Amador et al., 2001; Azevedo et al., 2001;Stone et al., 2003). The ubiquitination of proteins involves three enzymes: the E1 ubiquitin-activating enzyme, which forms a thioester intermediate with ubiquitin; the E2 ubiquitin-conjugating enzyme, which receives the ubiquitin molecule from the E1 enzyme; and the E3 ubiquitin ligase, which facilitates t...

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

Self-incompatibility, the rejection of self pollen, is the most widespread mechanism by which flowering plants prevent inbreeding. In Brassica, the S receptor kinase (SRK) has been implicated in the self-incompatibility response, but the molecular mechanisms involving SRK are unknown. One putative downstream effector for SRK is ARC1, a protein that binds to the SRK kinase domain. Here it is shown that suppression of ARC1 messenger RNA levels in the self-incompatible Brassica napus W1 line is correlated with a partial breakdown of self-incompatibility, resulting in seed production. This provides strong evidence that ARC1 is a positive effector of the Brassica self-incompatibility response.