Arabidopsis Skp1-like Research Articles

Adaptive and degenerative evolution of the S-Phase Kinase-Associated Protein 1-Like family in Arabidopsis thaliana.

Genome sequencing has uncovered tremendous sequence variation within and between species. In plants, in addition to large variations in genome size, a great deal of sequence polymorphism is also evident in several large multi-gene families, including those involved in the ubiquitin-26S proteasome protein degradation system. However, the biological function of this sequence variation is yet not clear. In this work, we explicitly demonstrated a single origin of retroposed Arabidopsis Skp1-Like (ASK) genes using an improved phylogenetic analysis. Taking advantage of the 1,001 genomes project, we here provide several lines of polymorphism evidence showing both adaptive and degenerative evolutionary processes in ASK genes. Yeast two-hybrid quantitative interaction assays further suggested that recent neutral changes in the ASK2 coding sequence weakened its interactions with some F-box proteins. The trend that highly polymorphic upstream regions of ASK1 yield high levels of expression implied negative expression regulation of ASK1 by an as-yet-unknown transcriptional suppression mechanism, which may contribute to the polymorphic roles of Skp1-CUL1-F-box complexes. Taken together, this study provides new evolutionary evidence to guide future functional genomic studies of SCF-mediated protein ubiquitylation.

The Arabidopsis F-box E3 ligase RIFP1 plays a negative role in abscisic acid signalling by facilitating ABA receptor RCAR3 degradation.

The phytohormone abscisic acid (ABA) plays a vital role in plant growth and development. The function of ABA is mediated by a group of newly discovered ABA receptors, named PYRABACTIN RESISTANCE 1/PYR-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORs (PYR1/PYLs/RCARs). Here, we report that an Arabidopsis thaliana F-box protein RCAR3 INTERACTING F-BOX PROTEIN 1 (RIFP1) interacts with ABA receptor (RCAR3) and SCF E3 ligase complex subunits Arabidopsis SKP1-LIKE PROTEINs (ASKs) in vitro and in vivo. The rifp1 mutant plants displayed increased ABA-mediated inhibition of seed germination and water loss of detached leaves, while the overexpression of RIFP1 in Arabidopsis led to plants being insensitive to ABA. Meanwhile, the rifp1 mutant plants showed greater tolerance to water deficit. In addition, the RCAR3 protein level was more stable in the rifp1 mutant plants than in the wild-type plants, indicating that RIFP1 facilitates the proteasome degradation of RCAR3. Accordingly, the loss of RIFP1 increased the transcript levels of several ABA-responsive genes. Taken together, these data indicate that RIFP1 plays a negative role in the RCAR3-mediated ABA signalling pathway and likely functions as an adaptor subunit of the SCF ubiquitin ligase complex to regulate ABA receptor RCAR3 stability.

Characterization of Arabidopsis Tubby-like proteins and redundant function of AtTLP3 and AtTLP9 in plant response to ABA and osmotic stress.

Tubby and Tubby-like proteins (TLPs) play essential roles in the development and function of mammal neuronal cells. In addition to the conserved carboxyl (C)-terminal Tubby domain, which is required for their plasma membrane (PM) tethering, plant TLPs also possess an amino (N)-terminal F-box domain to interact with specific Arabidopsis Skp1-like (ASK) proteins as functional SCF-type E3 ligases. Here, we report the molecular characterization of Arabidopsis TLPs (AtTLPs). β-Glucuronidase staining showed overlapped but distinct expression patterns of AtTLPs in Arabidopsis. Yeast two-hybrid assays further revealed that AtTLP1, AtTLP3, AtTLP6, AtTLP7, AtTLP9, AtTLP10 and AtTLP11 all interacted with specific ASKs, but AtTLP2, AtTLP5 and AtTLP8 did not. Subcellular localization observations in both Arabidopsis protoplasts and tobacco pollen tubes indicated that all GFP-AtTLP fusion proteins, except GFP-AtTLP8 which lacks the conserved phosphatidylinositol 4,5-bisphosphate binding sites, were targeted to the PM. Detailed studies on AtTLP3 demonstrated that AtTLP3 is a PM-tethered PIP2 binding protein which functions redundantly with AtTLP9 in abscisic acid (ABA)- and osmotic stress-mediated seed germination. Our results suggest that AtTLPs possibly work in multiple physiological and developmental processes in Arabidopsis, and AtTLP3 is also involved in ABA signaling pathway like AtTLP9 during seed germination and early seedling growth.

A Comprehensive Analysis of Interaction and Localization of Arabidopsis SKP1-LIKE (ASK) and F-Box (FBX) Proteins

F-Box (FBX) proteins are encoded by a multigene family present in major lineages of eukaryotes. A number of FBX proteins are shown to be subunits of SCF complex, a type of E3 ligases composed of SKP1, CULLIN, FBX and RBX1 proteins. The Arabidopsis SKP-LIKE (ASK) proteins are also members of a family and some of them interact with FBX proteins directly. To clarify how FBX and ASK proteins combine, we carried out a large-scale interaction analysis between FBX and ASK proteins using yeast two-hybrid assay (Y2H) in Arabidopsis thaliana. FBX proteins randomly chosen from those proteins that interacted with more than one ASK protein were further analyzed for their subcellular localization and in vivo interaction with ASK proteins. Furthermore, the expression profiles of FBX and ASK genes were compared. This work reveals that FBX proteins had a preference for interacting with ASK proteins depending on the domains they contain such as the FBX-associated (FBA) domain, the Kelch domain and leucine rich repeat (LRR). In addition, it was found that a single FBX protein could form multiple SCF complexes by interacting with several ASK proteins in many cases. Furthermore, it was suggested that the variation of SCF complexes were especially abundant in tissues related to male gametophyte and seed development. More than half of the FBX proteins studied did not interact with any of the ASK proteins, implying the necessity for certain regulations for their interaction in vivo and/or distinct roles from subunits of the SCF complex.

An F‐box gene, CPR30, functions as a negative regulator of the defense response in Arabidopsis

Arabidopsis gain-of-resistance mutants, which show HR-like lesion formation and SAR-like constitutive defense responses, were used well as tools to unravel the plant defense mechanisms. We have identified a novel mutant, designated constitutive expresser of PR genes 30 (cpr30), that exhibited dwarf morphology, constitutive resistance to the bacterial pathogen Pseudomonas syringae and the dramatic induction of defense-response gene expression. The cpr30-conferred growth defect morphology and defense responses are dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), and NONRACE-SPECIFIC DISEASE RESISTANCE 1 (NDR1). Further studies demonstrated that salicylic acid (SA) could partially account for the cpr30-conferred constitutive PR1 gene expression, but not for the growth defect, and that the cpr30-conferred defense responses were NPR1 independent. We observed a widespread expression of CPR30 throughout the plant, and a localization of CPR30-GFP fusion protein in the cytoplasm and nucleus. As an F-box protein, CPR30 could interact with multiple Arabidopsis-SKP1-like (ASK) proteins in vivo. Co-localization of CPR30 and ASK1 or ASK2 was observed in Arabidopsis protoplasts. Based on these results, we conclude that CPR30, a novel negative regulator, regulates both SA-dependent and SA-independent defense signaling, most likely through the ubiquitin-proteasome pathway in Arabidopsis.

Gene expression, localization, and protein–protein interaction of Arabidopsis SKP1-like (ASK) 20A and 20B

Arabidopsis SKP1-like 20 (ASK20) has two translational products, ASK20A and ASK20B, as a result of alternative splicing. Both ASK20A and ASK20B mRNAs were detected in every organ examined, and only small differences in their accumulation levels were observed in plants exposed to abiotic stress or treated with growth regulators. Transgenic plants possessing ASK20 promoter: GUS ( β-glucuronidase) displayed GUS activity in the root, leaf, flower, and seed, in agreement with reverse transcriptase-polymerase chain reaction (RT-PCR) results. Transgenic plants possessing ASK20 promoter: ASK20B-GUS showed a pattern of GUS activity similar to that of ASK20 promoter: GUS plants, whereas transgenic plants possessing ASK20 promoter: ASK20A-GUS displayed little GUS activity in the root vein, cotyledon, sepal, and stamen. GUS activity of the transiently expressed ASK20A-GUS and ASK20B-GUS as well as green fluorescent protein (GFP) signals for ASK20A-GFP and ASK20B-GFP in transgenic plants were localized in the cytosol. In a yeast two-hybrid system, both ASK20A and ASK20B interacted with two F-box proteins (At3g61590 and At5g39250). Neither ASK20A nor ASK20B interacted with CUL1 (cullin1) in the yeast two- or three-hybrid system, and little interaction of ASK20 proteins with CUL1 was detected in a pull-down assay.

Patterns of gene duplication in the plant SKP1 gene family in angiosperms: evidence for multiple mechanisms of rapid gene birth

Gene duplication plays important roles in organismal evolution, because duplicate genes provide raw materials for the evolution of mechanisms controlling physiological and/or morphological novelties. Gene duplication can occur via several mechanisms, including segmental duplication, tandem duplication and retroposition. Although segmental and tandem duplications have been found to be important for the expansion of a number of multigene families, the contribution of retroposition is not clear. Here we show that plant SKP1 genes have evolved by multiple duplication events from a single ancestral copy in the most recent common ancestor (MRCA) of eudicots and monocots, resulting in 19 ASK (Arabidopsis SKP1-like) and 28 OSK (Oryza SKP1-like) genes. The estimated birth rates are more than ten times the average rate of gene duplication, and are even higher than that of other rapidly duplicating plant genes, such as type I MADS box genes, R genes, and genes encoding receptor-like kinases. Further analyses suggest that a relatively large proportion of the duplication events may be explained by tandem duplication, but few, if any, are likely to be due to segmental duplication. In addition, by mapping the gain/loss of a specific intron on gene phylogenies, and by searching for the features that characterize retrogenes/retrosequences, we show that retroposition is an important mechanism for expansion of the plant SKP1 gene family. Specifically, we propose that two and three ancient retroposition events occurred in lineages leading to Arabidopsis and rice, respectively, followed by repeated tandem duplications and chromosome rearrangements. Our study represents a thorough investigation showing that retroposition can play an important role in the evolution of a plant gene family whose members do not encode mobile elements.

Proteomic identification of potential target proteins regulated by an ASK1-mediated proteolysis pathway

The ASK1 (ARABIDOPSIS SKP1-LIKE) protein is a critical component of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase complexes that recruit target proteins for degradation by the 26S proteosome. To investigate proteins that are affected by the ASK1-mediated proteolysis pathway in Arabidopsis flowers, we compared the proteomes of the Arabidopsis wild type and ask1 mutant flower buds using two-dimensional electrophoresis (2-DE). Ten protein spots with higher or lower abundance in the ask1 mutant flowers compared to wild type flowers were excised and subjected to further mass spectrometry (MS) analysis. The results showed that they were proteins involved in photomorphogenesis, circadian oscillation, post-translation process, stress-responses and cell expansion or elongation, suggesting that those processes were affected in the ask1 mutant. The transcript levels of these genes were also compared based on the Affymetrix gene chip microarray data. No significant difference was observed for most of the genes, suggesting that the proteins with elevated levels of accumulation in the ask1 mutant could be candidate targets regulated by an ASK1-mediated proteolysis pathway. These results help to elucidate the pleiotropic functions of ASK1 in Arabidopsis developmental processes and also demonstrate the importance and necessity of studying protein levels with respect to gene functions.

Functional analysis of EID1, an F‐box protein involved in phytochrome A‐dependent light signal transduction

Empfindlicher im Dunkelroten Licht 1 (EID1) is an F-box protein that functions as a negative regulator in phytochrome A (phyA)-specific light signalling. F-box proteins are components of SCF ubiquitin ligase complexes that target proteins for degradation in the proteasome. Here we present further characterization of EID1 at the expression level, and show that it regulates photomorphogenesis in seedlings, rosette leaf development and flowering. Data on transcript expression patterns indicate that EID1 is expressed during all stages of Arabidopsis development and exhibits no light response. Microscope studies demonstrate that EID1 is localized to the nucleus, where it can form speckles under continuous far-red light that resemble clastosomes. To characterize the composition and formation of SCF(EID1) complexes further, we used two-hybrid and bridge assays in yeast and in planta. EID1 interacts specifically with several Arabidopsis Skp1-like (ASK) proteins and Cullin1 to form stable dimeric and trimeric complexes. Our results support a two-step association process in which the F-box protein binds first to the ASK adaptor, forming a unit which then associates with the catalytic core of the SCF complex. Finally, our data indicate that the EID1 target interaction domain is composed of two independent modules.

Cloning and expression analysis of TSK1, a wheat SKP1 homologue, and functional comparison with Arabidopsis ASK1 in male meiosis and auxin signalling

Plants possess multiple homologues of the SKP1 gene encoding an essential subunit of the SCF ubiquitin ligases, but only ASK1 (Arabidopsis SKP1-like 1) and ASK2 have been characterised genetically. In addition, little is known about the function of SKP1 homologues in monocots. Here we report on a winter wheat homologue of SKP1 named TSK1 (Triticum aestivum SKP1-like 1). Expression analyses revealed that it was expressed predominantly in young roots and floral buds. RNA in situ hybridisation showed that it was expressed in the shoot apical meristem (SAM) and anthers, especially the tapetum and microsporocytes at the time of meiosis. It was also expressed in almost the entire meristematic and elongation zones of the root. These observations indicated that TSK1 might function in dividing cells. The Arabidopsis ask1-1 mutant with overexpressed TSK1 driven by the CaMV 35S promoter exhibited partial fertility, suggesting that TSK1 could partially restore function in meiosis to the ask1-1 mutant. In addition, overexpression of TSK1 in wild type Arabidopsis resulted in changes in auxin responses and auxin-related phenotypes, consistent with a role of ASK1 in Arabidopsis auxin response. These results suggest possible functional conservation between TSK1 and ASK1.

Genome-wide analysis of S-Locus F-box-like genes in Arabidopsis thaliana

The Antirrhinum S-locus F-box gene, AhSLF-S2, has been shown to determine the pollen function of S-RNase-mediated self-incompatibility (SI). Its initial identification led to the discovery of a large family of plant-specific F-box proteins, named the SLF (S-Locus F-box) family, including members from species with or without S-RNase SI system. To investigate the evolution and function of its family members in Arabidopsis, we first identified 92 Arabidopsis F-box proteins related to AhSLF-S2, referred to as AtSFL (S-locus F-box-like) in this report. Phylogenetic analyses with family members from several plant species revealed that they could be classified into five subgroups, and the SLF genes appeared to have had a monophyletic origin. Yeast two-hybrid analyses showed that most AtSFL proteins could interact with one or more ASK (Arabidopsis Skp1-like) proteins, a component of the SCF (Skp1/Cullin or CDC53/F-box) complex, suggesting that AtSFLs may function in the process of ubiquitin/26S proteasome-mediated proteolysis. Transcript analysis found that most of AtSFL genes are expressed ubiquitously and only three of them (AtSFL61, 79 and 85) displayed a tissue-specific pattern. In consistent, phenotypic observations for T-DNA insertion lines of 37 AtSFL genes revealed that most of them are functionally redundant, but inactivation of two AtSFL genes (AtSFL 61 and 70) appears to have caused developmental defects in embryo or female gametophyte. Our results show that a diversified expression and functional pattern are associated with AtSFL genes, indicating that they play important roles in various biological processes in Arabidopsis.

Members of the Arabidopsis-SKP1-like gene family exhibit a variety of expression patterns and may play diverse roles in Arabidopsis.

Ubiquitin-mediated proteolysis by the proteasome is a critical regulatory mechanism controlling many biological processes. In particular, SKP1, cullin/CDC53, F-box protein (SCF) complexes play important roles in selecting substrates for proteolysis by facilitating the ligation of ubiquitin to specific proteins. In plants, SCF complexes have been found to regulate auxin responses and jasmonate signaling and may be involved in several other processes, such as flower development, circadian clock, and gibberellin signaling. Although 21 Skp1-related genes, called Arabidopsis-SKP1-like (ASK), have been uncovered in the Arabidopsis genome, ASK1 is the only gene that has been analyzed genetically. As a first step toward understanding their functions, we tested for expression of 20 ASK genes using reverse transcription-polymerase chain reaction experiments. Also, we examined the expression patterns of 11 ASK genes by in situ hybridizations. The ASK genes exhibit a spectrum of expression levels and patterns, with a large subset showing expression in the flower and/or fruit. In addition, the ASK genes that have similar sequences tend to have similar expression patterns. On the basis of the expression results, we selectively suppressed the expression of a few ASK genes using RNA interference. Compared with the ask1 mutant, the strong ASK1 RNA interference (RNAi) line exhibited similar or enhanced phenotypes in both vegetative and floral development, whereas ASK11 RNAi plants had normal vegetative growth but mild defects in flower development. The diverse expression patterns and distinct defects observed in RNAi plants suggest that the ASK gene family may collectively perform a range of functions and may regulate different developmental and physiological processes.

Protein interaction analysis of SCF ubiquitin E3 ligase subunits from Arabidopsis.

Ubiquitin E3 ligases are a diverse family of protein complexes that mediate the ubiquitination and subsequent proteolytic turnover of proteins in a highly specific manner. Among the several classes of ubiquitin E3 ligases, the Skp1-Cullin-F-box (SCF) class is generally comprised of three 'core' subunits: Skp1 and Cullin, plus at least one F-box protein (FBP) subunit that imparts specificity for the ubiquitination of selected target proteins. Recent genetic and biochemical evidence in Arabidopsis thaliana suggests that post-translational turnover of proteins mediated by SCF complexes is important for the regulation of diverse developmental and environmental response pathways. In this report, we extend upon a previous annotation of the Arabidopsis Skp1-like (ASK) and FBP gene families to include the Cullin family of proteins. Analysis of the protein interaction profiles involving the products of all three gene families suggests a functional distinction between ASK proteins in that selected members of the protein family interact generally while others interact more specifically with members of the F-box protein family. Analysis of the interaction of Cullins with FBPs indicates that CUL1 and CUL2, but not CUL3A, persist as components of selected SCF complexes, suggesting some degree of functional specialization for these proteins. Yeast two-hybrid analyses also revealed binary protein interactions between selected members of the FBP family in Arabidopsis. These and related results are discussed in terms of their implications for subunit composition, stoichiometry and functional diversity of SCF complexes in Arabidopsis.

The Arabidopsis SKP1-like genes present a spectrum of expression profiles.

The yeast Skp1 protein is a component of the SCF complex, an E3 enzyme involved in the specific protein degradation pathway via ubiquitination. Skp1 binds to F-box proteins to trigger specific recognition of proteins targeted for degradation. SKP1-like genes have been found in a variety of eukaryotes including yeast, man, Caenorhabditis elegans and Arabidopsis thaliana. The Arabidopsis genome contains 20 SKP1-like genes called ASK (for Arabidopsis SKP1-like), among which only ASK1 has been characterized in detail. The analysis of the expression pattern of the ASK genes in Arabidopsis should provide key information for the understanding of the biological role of this family in protein degradation and in different cellular mechanisms. In this paper, we describe the expression profiles of 19 ASK promoter-GUS fusions in stable transformants of Arabidopsis, with a special emphasis on floral organ development. Four ASK promoters did not show any detectable expression in either inflorescences or seedlings. Our results on the ASK1 expression profile are consistent with previous reports. Several ASK promoters show clear tissue-specific expression (for instance in the connective of anthers or in the embryo). We also found that almost half (9/19) of ASK promoters direct a post-meiotic expression in the male gametophyte. Tight regulation of the expression of this gene family indicates a crucial role of the ubiquitin degradation pathway during development, particularly during male gametophyte development.