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.     

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.     

Characterization of three novel members of the Arabidopsis SHAGGY-related protein kinase (ASK) multigene family

In this paper we report the characterization of three novel members of the Arabidopsis shaggy-related protein kinase (ASK) multigene family, named ASKdzeta (ASK), ASKetha (ASK) and ASKiota (ASK). The proteins encoded by the ASK genes share a highly conserved catalytic protein kinase domain and show about 70% identity to SHAGGY (SGG) and glycogen synthase kinase-3 (GSK-3) from Drosophila and rat respectively. SGG is an ubiquitous intracellular component of the wingless signalling pathway that establishes cell fate and/or pattern formation in Drosophila. At least ten different ASK genes are expected to be present per haploid genome of A. thaliana. Different amino- and carboxy-terminal extensions distinguish different ASK family members. Five ASK gene sequences were analysed and shown to be present as single-copy genes in the Arabidopsis genome. A comparison based on the highly conserved catalytic domain sequences of all known sequences of the GSK-3 subfamily of protein kinases demonstrated a clear distinction between the plant and the animal kinases. Furthermore, we established the presence of at least three distinct groups of plant homologues of SGG/GSK-3. These different groups probably reflect biochemical and/or biological properties of these kinases. The differential expression patterns of five ASK genes were accessed by northern and in situ hybridization experiments using gene-specific probes. While ASK is expressed in the whole embryo during its development, ASK expression is limited to the suspensor cells. No signal was detected for ASK, ASK and ASK in developing embryos.     

Functional Analysis of the Brassica napus L. Phytoene Synthase (PSY) Gene Family

Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three “Arabidopsis-like” subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.     

Comprehensive Evolutionary and Expression Analysis of FCS-Like Zinc finger Gene Family Yields Insights into Their Origin,Expansion and Divergence

Plant evolution is characterized by frequent genome duplication events. Expansion of habitat resulted in the origin of many novel genes and genome duplication events which in turn resulted in the expansion of many regulatory gene families. The plant-specific FCS-Like Zinc finger (FLZ) gene family is characterized by the presence of a FCS-Like Zinc finger (FLZ) domain which mediates the protein-protein interaction. In this study, we identified that the expansion of FLZ gene family size in different species is correlated with ancestral and lineage-specific whole genome duplication events. The subsequent gene loss found to have a greater role in determining the size of this gene family in many species. However, genomic block duplications played the significant role in the expansion of FLZ gene family in some species. Comparison of Arabidopsis thaliana and Oryza sativa FLZ gene family revealed monocot and dicot specific evolutionary trends. The FLZ genes were found to be under high purifying selection. The spatiotemporal expression analyses of Arabidopsis thaliana FLZ gene family revealed that majority of the members are highly expressed in reproductive organs. FLZ genes were also found to be highly expressed during vegetative-to-reproductive phase transition which is correlated with the proposed role of this gene family in sugar signaling. The comparison of sequence, structural and expression features of duplicated genes identified lineage-specific redundancy and divergence. This extensive evolutionary analysis and expression analysis of Arabidopsis thaliana FLZ genes will pave the way for further functional analysis of FLZ genes.     

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.     

Cohesion Group Approach for Evolutionary Analysis of Aspartokinase,an Enzyme That Feeds a Branched Network of Many Biochemical Pathways

Summary: Aspartokinase (Ask) exists within a variable network that supports the synthesis of 9 amino acids and a number of other important metabolites. Lysine, isoleucine, aromatic amino acids, and dipicolinate may arise from the ASK network or from alternative pathways. Ask proteins were subjected to cohesion group analysis, a methodology that sorts a given protein assemblage into groups in which evolutionary continuity is assured. Two subhomology divisions, ASK_α and ASK_β, have been recognized. The ASK_α subhomology division is the most ancient, being widely distributed throughout the Archaea and Eukarya and in some Bacteria. Within an indel region of about 75 amino acids near the N terminus, ASK_β sequences differ from ASK_α sequences by the possession of a proposed ancient deletion. ASK_β sequences are present in most Bacteria and usually exhibit an in-frame internal translational start site that can generate a small Ask subunit that is identical to the C-terminal portion of the larger subunit of a heterodimeric unit. Particularly novel are ask genes embedded in gene contexts that imply specialization for ectoine (osmotic agent) or aromatic amino acids. The cohesion group approach is well suited for the easy recognition of relatively recent lateral gene transfer (LGT) events, and many examples of these are described. Given the current density of genome representation for Proteobacteria, it is possible to reconstruct more ancient landmark LGT events. Thus, a plausible scenario in which the three well-studied and iconic Ask homologs of Escherichia coli are not within the vertical genealogy of Gammaproteobacteria, but rather originated via LGT from a Bacteroidetes donor, is supported.     

Isolation and Characterization of a Ferredoxin Gene from Arabidopsis thaliana

We report the cloning and characterization of an Arabidopsis thaliana (L.) Heynh. (Columbia ecotype) ferredoxin gene (Fed A). Sequence analysis of a genomic clone shows an intron-free, 444-base pair open reading frame which encodes a 96 amino acid mature ferredoxin polypeptide preceded by a 52 amino acid transit peptide. Comparison with other plant ferredoxin proteins suggests that Fed A encodes a leaf ferredoxin. Genomic Southern blot analysis indicates the presence of a second, weakly related gene, consistent with other reports of at least two ferredoxins in plants. The Fed A gene promoter contains two regions, ACGCCACGTGGTAGATAGGATT (G-I box) and CCACGCCATTTCCACAAGC (CCAC box), which are strongly conserved in both sequence and position between the Arabidopsis and pea ferredoxin genes. Similarities with other better characterized plant promoter elements are also discussed.     

Overexpression of a Soybean Ariadne-Like Ubiquitin Ligase Gene GmARI1 Enhances Aluminum Tolerance in Arabidopsis

Ariadne (ARI) subfamily of RBR (Ring Between Ring fingers) proteins have been found as a group of putative E3 ubiquitin ligases containing RING (Really Interesting New Gene) finger domains in fruitfly, mouse, human and Arabidopsis. Recent studies showed several RING-type E3 ubiquitin ligases play important roles in plant response to abiotic stresses, but the function of ARI in plants is largely unknown. In this study, an ariadne-like E3 ubiquitin ligase gene was isolated from soybean, Glycine max (L.) Merr., and designated as GmARI1. It encodes a predicted protein of 586 amino acids with a RBR supra-domain. Subcellular localization studies using Arabidopsis protoplast cells indicated GmARI protein was located in nucleus. The expression of GmARI1 in soybean roots was induced as early as 2–4 h after simulated stress treatments such as aluminum, which coincided with the fact of aluminum toxicity firstly and mainly acting on plant roots. In vitro ubiquitination assay showed GmARI1 protein has E3 ligase activity. Overexpression of GmARI1 significantly enhanced the aluminum tolerance of transgenic Arabidopsis. These findings suggest that GmARI1 encodes a RBR type E3 ligase, which may play important roles in plant tolerance to aluminum stress.     

拟南芥ASK基因研究进展拟南芥ASK基因研究进展拟南芥ASK基因研究进展拟南芥ASK基因研究进展拟南芥ASK基因研究进展

泛素-蛋白酶体系统（ubiquitin-proteasome system, UPS）是广泛存在于真核生物中的一种重要的蛋白降解系统。拟南芥ASK （Arabidopsis SKP1-LIKE）基因编码拟南芥E3连接酶SCF复合物的一个亚蛋白, 在拟南芥SCF复合物中起到连接器的作用。近年来, 人们对ASK基因及其同源基因进行了很多表达规律、基因功能方面的研究。本文从ASK基因表达方式、对生理发育过程的调节、与F-box相互作用及ASK基因的进化方式4个方面对这些进展进行总结。已有的研究结果表明, ASK基因在拟南芥中广泛地表达并表现出各自不同的表达水平和表达方式, 它们在很多发育和生理过程中起到重要作用。     

Regulation of flower development in Arabidopsis by SCF complexes

SCF complexes are the largest and best studied family of E3 ubiquitin protein ligases that facilitate the ubiquitylation of proteins targeted for degradation. The SCF core components Skp1, Cul1, and Rbx1 serve in multiple SCF complexes involving different substrate-specific F-box proteins that are involved in diverse processes including cell cycle and development. In Arabidopsis, mutations in the F-box gene UNUSUAL FLORAL ORGANS (UFO) result in a number of defects in flower development. However, functions of the core components Cul1 and Rbx1 in flower development are poorly understood. In this study we analyzed floral phenotypes caused by altering function of Cul1 or Rbx1, as well as the effects of mutations in ASK1 and ASK2. Plants homozygous for a point mutation in the AtCUL1 gene showed reduced floral organ number and several defects in each of the four whorls. Similarly, plants with reduced AtRbx1 expression due to RNA interference also exhibited floral morphological defects. In addition, compared to the ask1 mutant, plants homozygous for ask1 and heterozygous for ask2 displayed enhanced reduction of B function, as well as other novel defects of flower development, including carpelloid sepals and an inhibition of petal development. Genetic analyses demonstrate that AGAMOUS (AG) is required for the novel phenotypes observed in the first and second whorls. Furthermore, the genetic interaction between UFO and AtCUL1 supports the idea that UFO regulates multiple aspects of flower development as a part of SCF complexes. These results suggest that SCF complexes regulate several aspects of floral development in Arabidopsis.     

Structural,Expression and Interaction Analysis of Rice SKP1-Like Genes

The degradation of proteins by the 26S proteasome is initiated by protein polyubiquitination mediated by a three-step cascade. The specific ubiquitination of different target proteins is mediated by different classes of E3 ubiquitin ligases, among which the best known are Skp1-Cullin-F-box complexes. Whereas protists, fungi and some vertebrates have a single SKP1 gene, many animal and plant species possess multiple SKP1 homologues. In this paper, we report on the structure, phylogeny and expression of the complete set of rice SKP1 genes (OSKs, Oryza sativa SKP1-like genes). Our analyses indicated that OSK1 and OSK20 belong to a class of SKP1 genes that contain one intron at a conserved position and are highly expressed. In addition, our yeast two-hybrid results revealed that OSK proteins display a differing ability to interact with F-box proteins. However, OSK1 and OSK20 seemed to interact with most of the nine F-box proteins tested. We suggest that rice OSK1 and OSK20 are likely to have functions similar to the Arabidopsis ASK1 and ASK2 genes.     

DNA Sequence Analysis of a Complementary DNA for Cold-Regulated Arabidopsis Gene cor15 and Characterization of the COR 15 Polypeptide

Previous studies have indicated that changes in gene expression occur in Arabidopsis thaliana L. (Heyn) during cold acclimation and that certain of the cor (cold-regulated) genes encode polypeptides that share the unusual property of remaining soluble upon boiling in aqueous solution. Here, we identify a cDNA clone for a cold-regulated gene encoding one of the “boiling-stable” polypeptides, COR15. DNA sequence analysis indicated that the gene, designated cor15, encodes a 14.7-kilodalton hydrophilic polypeptide having an N-terminal amino acid sequence that closely resembles transit peptides that target proteins to the stromal compartment of chloroplasts. Immunological studies indicated that COR15 is processed in vivo and that the mature polypeptide, COR 15m, is present in the soluble fraction of chloroplasts. Possible functions of COR 15m are discussed.     

Functional divergence in the Arabidopsis LOB-domain gene family

The Arabidopsis LOB-domain (LBD) gene family is composed by 43 members divided in two classes based on amino acid conservation within the LOB-domain. The LOB domain is known to be responsible for DNA binding and protein-protein interactions. There is very little functional information available for most genes in the LBD family and many lbd single mutants do not exhibit conspicuous phenotypes. One plausible explanation for the limited loss-of-function phenotypes observed in this family is that LBD genes exhibit significant functional redundancy. Here we discuss an example of one phylogenetic subgroup of the LBD family, in which genes that are closely related based on phylogeny exhibit distinctly different expression patterns and do not have overlapping functions. We discuss the challenges of using phylogenetic analyses to predict redundancy in gene families.     

Functional Conservation of MIKC*-Type MADS Box Genes in Arabidopsis and Rice Pollen Maturation

There are two groups of MADS intervening keratin-like and C-terminal (MIKC)-type MADS box genes, MIKC^C type and MIKC* type. In seed plants, the MIKC^C type shows considerable diversity, but the MIKC* type has only two subgroups, P- and S-clade, which show conserved expression in the gametophyte. To examine the functional conservation of MIKC*-type genes, we characterized all three rice (Oryza sativa) MIKC*-type genes. All three genes are specifically expressed late in pollen development. The single knockdown or knockout lines, respectively, of the S-clade MADS62 and MADS63 did not show a mutant phenotype, but lines in which both S-clade genes were affected showed severe defects in pollen maturation and germination, as did knockdown lines of MADS68, the only P-clade gene in rice. The rice MIKC*-type proteins form strong heterodimeric complexes solely with partners from the other subclade; these complexes specifically bind to N10-type C-A-rich-G-boxes in vitro and regulate downstream gene expression by binding to N10-type promoter motifs. The rice MIKC* genes have a much lower degree of functional redundancy than the Arabidopsis thaliana MIKC* genes. Nevertheless, our data indicate that the function of heterodimeric MIKC*-type protein complexes in pollen development has been conserved since the divergence of monocots and eudicots, roughly 150 million years ago.     

The Drosophila Gene abnormal spindle Encodes a Novel Microtubule-associated Protein That Associates with the Polar Regions of the Mitotic Spindle

abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp¹ directs the synthesis of a COOH terminally truncated or internally deleted peptide of ~124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH₂-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34^cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.