Arabidopsis Skp1-like 1 Research Articles

Arabidopsis F-BOX STRESS INDUCED 4 is required to repress excessive divisions in stomatal development.

During the terminal stage of stomatal development, the R2R3-MYB transcription factors FOUR LIPS (FLP/MYB124) and MYB88 limit guard mother cell division by repressing the transcript levels of multiple cell-cycle genes. In Arabidopsis thaliana possessing the weak allele flp-1, an extra guard mother cell division results in two stomata having direct contact. Here, we identified an ethylmethane sulfonate-mutagenized mutant, flp-1 xs01c, which exhibited more severe defects than flp-1 alone, producing giant tumor-like cell clusters. XS01C, encoding F-BOX STRESS-INDUCED 4 (FBS4), is preferentially expressed in epidermal stomatal precursor cells. Overexpressing FBS4 rescued the defective stomatal phenotypes of flp-1 xs01c and flp-1 mutants. The deletion or substitution of a conserved residue (Proline166) within the F-box domain of FBS4 abolished or reduced, respectively, its interaction with Arabidopsis Skp1-Like1 (ASK1), the core subunit of the Skp1/Cullin/F-box E3 ubiquitin ligase complex. Furthermore, the FBS4 protein physically interacted with CYCA2;3 and induced its degradation through the ubiquitin-26S proteasome pathway. Thus, in addition to the known transcriptional pathway, the terminal symmetric division in stomatal development is ensured at the post-translational level, such as through the ubiquitination of target proteins recognized by the stomatal lineage F-box protein FBS4.

Diverse Evolution in 111 Plant Genomes Reveals Purifying and Dosage Balancing Selection Models for F-Box Genes.

The F-box proteins function as substrate receptors to determine the specificity of Skp1-Cul1-F-box ubiquitin ligases. Genomic studies revealed large and diverse sizes of the F-box gene superfamily across plant species. Our previous studies suggested that the plant F-box gene superfamily is under genomic drift evolution promoted by epigenomic programming. However, how the size of the superfamily drifts across plant genomes is currently unknown. Through a large-scale genomic and phylogenetic comparison of the F-box gene superfamily covering 110 green plants and one red algal species, I discovered four distinct groups of plant F-box genes with diverse evolutionary processes. While the members in Clusters 1 and 2 are species/lineage-specific, those in Clusters 3 and 4 are present in over 46 plant genomes. Statistical modeling suggests that F-box genes from the former two groups are skewed toward fewer species and more paralogs compared to those of the latter two groups whose presence frequency and sizes in plant genomes follow a random statistical model. The enrichment of known Arabidopsis F-box genes in Clusters 3 and 4, along with comprehensive biochemical evidence showing that Arabidopsis members in Cluster 4 interact with the Arabidopsis Skp1-like 1 (ASK1), demonstrates over-representation of active F-box genes in these two groups. Collectively, I propose purifying and dosage balancing selection models to explain the lineage/species-specific duplications and expansions of F-box genes in plant genomes. The purifying selection model suggests that most, if not all, lineage/species-specific F-box genes are detrimental and are thus kept at low frequencies in plant genomes.

Generation of a fertile ask1 mutant uncovers a comprehensive set of SCF-mediated intracellular functions.

Many eukaryotic intracellular processes employ protein ubiquitylation by ubiquitin E3 ligases for functional regulation or protein quality control. In plants, the multi-subunit Skp1-Cullin1-F-box (SCF) complexes compose the largest group of E3 ligases whose specificity is determined by a diverse array of F-box proteins. Although both sequence divergence and polymorphism of F-box genes well support a broad spectrum of SCF functions, experimental evidence is scarce due to the low number of identified SCF substrates. Taking advantage of the bridge role of Skp1 between F-box and Cullin1 in the complex, we systematically analyzed the functional influence of a well-characterized Arabidopsis Skp1-Like1 (ASK1) Ds insertion allele, ask1, in different Arabidopsis accessions. Through 10 generations of backcrossing with Columbia-0 (Col-0), we partially rescued the fertility of this otherwise sterile ask1 allele in Landsberg erecta, thus providing experimental evidence showing the polymorphic roles of SCF complexes. This ask1 mutant produces twisted rosette leaves, a reduced number of petals, fewer viable pollen grains, and larger embryos and seeds compared to Col-0. RNA-Seq-based transcriptome analysis of ask1 uncovered a large spectrum of SCF functions, which is greater than a 10-fold increase compared with previous studies. We also identified its hyposensitive responses to auxin and abscisic acid treatments and enhanced far-red light/phyA-mediated photomorphogenesis. Such diverse roles are consistent with the 20-30% reduction of ubiquitylation events in ask1 estimated by immunoblotting analysis in this work. Collectively, we conclude that ASK1 is a predominant Skp1 protein in Arabidopsis and that the fertile ask1 mutant allowed us to uncover a comprehensive set of SCF functions.

Meiocyte-Specific and AtSPO11-1-Dependent Small RNAs and Their Association with Meiotic Gene Expression and Recombination.

Meiotic recombination ensures accurate chromosome segregation and results in genetic diversity in sexually reproducing eukaryotes. Over the last few decades, the genetic regulation of meiotic recombination has been extensively studied in many organisms. However, the role of endogenous meiocyte-specific small RNAs (ms-sRNAs; 21-24 nucleotide [nt]) and their involvement in meiotic recombination are unclear. Here, we sequenced the total small RNA (sRNA) and messenger RNA populations from meiocytes and leaves of wild type Arabidopsis (Arabidopsis thaliana) and meiocytes of spo11-1, a mutant defective in double-strand break formation, and we discovered 2,409 ms-sRNA clusters, 1,660 of which areSPORULATION 11-1 (AtSPO11-1)-dependent. Unlike mitotic small interfering RNAs that are enriched in intergenic regions and associated with gene silencing, ms-sRNAs are significantly enriched in genic regions and exhibit a positive correlation with genes that are preferentially expressed in meiocytes (i.e. Arabidopsis SKP1-LIKE1 and RAD51), in a fashion unrelated to DNA methylation. We also found that AtSPO11-1-dependent sRNAs have distinct characteristics compared with ms-sRNAs and tend to be associated with two known types of meiotic recombination hotspot motifs (i.e. CTT-repeat and A-rich motifs). These results reveal different meiotic and mitotic sRNA landscapes and provide new insights into how sRNAs relate to gene expression in meiocytes and meiotic recombination.

Regulation of SCFTIR1/AFBs E3 ligase assembly by S-nitrosylation of Arabidopsis SKP1-like1 impacts on auxin signaling

The F-box proteins (FBPs) TIR1/AFBs are the substrate recognition subunits of SKP1–cullin–F-box (SCF) ubiquitin ligase complexes and together with Aux/IAAs form the auxin co-receptor. Although tremendous knowledge on auxin perception and signaling has been gained in the last years, SCFTIR1/AFBs complex assembly and stabilization are emerging as new layers of regulation. Here, we investigated how nitric oxide (NO), through S-nitrosylation of ASK1 is involved in SCFTIR1/AFBs assembly. We demonstrate that ASK1 is S-nitrosylated and S-glutathionylated in cysteine (Cys) 37 and Cys118 residues in vitro. Both, in vitro and in vivo protein-protein interaction assays show that NO enhances ASK1 binding to CUL1 and TIR1/AFB2, required for SCFTIR1/AFB2 assembly. In addition, we demonstrate that Cys37 and Cys118 are essential residues for proper activation of auxin signaling pathway in planta. Phylogenetic analysis revealed that Cys37 residue is only conserved in SKP proteins in Angiosperms, suggesting that S-nitrosylation on Cys37 could represent an evolutionary adaption for SKP1 function in flowering plants. Collectively, these findings indicate that multiple events of redox modifications might be part of a fine-tuning regulation of SCFTIR1/AFBs for proper auxin signal transduction.

Identification and Functional Analysis of Two Cotton Orthologs of MAX2 Which Control Shoot Lateral Branching

Cotton (Gossypium spp.), as the most important fiber and oilseed crop in the world, is extremely important for the industry. However, due to its indeterminate growth habit and complex branching system, massive labor costs are needed for shoot apex removal and branch pruning during cotton production. Therefore, it is very important to explore branch-controlling genes and genetically modify the branch architecture of cotton. Strigolactones (SLs) are a novel class of plant hormone that inhibit the outgrowth of lateral branches. To elucidate the role of SLs in branch development of cotton, we cloned and characterized GhMAX2a and GhMAX2b from tetraploid upland cotton (Gossypium hirsutum), the orthologs of Arabidopsis MAX2, rice D3, and petunia RMS4. GhMAX2a/2b was ubiquitously expressed in all tested tissues of cotton, with relatively higher expression levels in leaves and lateral buds. Subcellular localization assay showed that the GhMAX2-GFP fusion protein localized to the nucleus. Both GhMAX2a and GhMAX2b can fully rescue the dwarfed and highly branched phenotypes of the Arabidopsis max2-1 mutant, indicating that GhMAX2s have conserved functions with that of AtMAX2. The cotton GhMAX2b interacted with Arabidopsis Skp1-like 1 (ASK1) proteins in vitro which was further confirmed in the Arabidopsis protoplasts using the co-immunoprecipitation assay, indicating that GhMAX2b probably functions through forming an SCF E3 complex with Skp and other proteins in the Arabidopsis. These results suggest that the cotton GhMAX2s encode functional MAX2 that can inhibit the shoot lateral branching. Further functional analysis of GhMAX2s in determining cotton branch architecture and yield is underway.

Proteomics and transcriptomics analyses of Arabidopsis floral buds uncover important functions of ARABIDOPSIS SKP1-LIKE1.

BackgroundThe ARABIDOPSIS SKP1-LIKE1 (ASK1) protein functions as a subunit of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. Previous genetic studies showed that ASK1 plays important roles in Arabidopsis flower development and male meiosis. However, the molecular impact of ASK1-containing SCF E3 ubiquitin ligases (ASK1-E3s) on the floral proteome and transcriptome is unknown.ResultsHere we identified proteins that are potentially regulated by ASK1-E3s by comparing floral bud proteomes of wild-type and the ask1 mutant plants. More than 200 proteins were detected in the ask1 mutant but not in wild-type and >300 were detected at higher levels in the ask1 mutant than in wild-type, but their RNA levels were not significantly different between wild-type and ask1 floral buds as shown by transcriptomics analysis, suggesting that they are likely regulated at the protein level by ASK1-E3s. Integrated analyses of floral proteomics and transcriptomics of ask1 and wild-type uncovered several potential aspects of ASK1-E3 functions, including regulation of transcription regulators, kinases, peptidases, and ribosomal proteins, with implications on possible mechanisms of ASK1-E3 functions in floral development.ConclusionsOur results suggested that ASK1-E3s play important roles in Arabidopsis protein degradation during flower development. This study opens up new possibilities for further functional studies of these candidate E3 substrates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0571-9) contains supplementary material, which is available to authorized users.

Distinct roles of FKF1, Gigantea, and Zeitlupe proteins in the regulation of Constans stability in Arabidopsis photoperiodic flowering.

Many plants measure changes in day length to synchronize their flowering time with appropriate seasons for maximum reproductive success. In Arabidopsis, the day-length-dependent regulation of Constans (CO) protein stability is crucial to induce flowering locus T (FT) expression for flowering in long days. The flavin-binding, KELCH repeat, F-box1 (FKF1) protein binds to CO protein specifically in the long-day afternoon and stabilizes it, although the mechanism remains unknown. Here we demonstrated that the FKF1-interacting proteins Gigantea (GI) and Zeitlupe (ZTL) are involved in CO stability regulation. First, our immunoprecipitation-mass spectrometry analysis of FKF1 revealed that FKF1 forms an S-phase kinase-associated protein 1 (Skp1)/Cullin(CUL)/F-box complex through interactions with Arabidopsis Skp1-like 1 (ASK1), ASK2, and CUL1 proteins and mainly interacts with GI protein in vivo. GI interacts with CO directly and indirectly through FKF1. Unexpectedly, the gi mutation increases the CO protein levels in the morning in long days. This gi-dependent destabilization of CO protein was cancelled by the fkf1 mutation. These results suggest that there are other factors likely influenced by both gi and fkf1 mutations that also control CO stability. We found that ZTL, which interacts with GI and FKF1, may be one such factor. ZTL also interacts with CO in vivo. The CO protein profile in the ztl mutant resembles that in the gi mutant, indicating that ZTL activity also may be changed in the gi mutant. Our findings suggest the presence of balanced regulation among FKF1, GI, and ZTL on CO stability regulation for the precise control of flowering time.

The role for CYCLIN A1;2/TARDY ASYNCHRONOUS MEIOSIS in differentiated cells in Arabidopsis

The Arabidopsis A1-type cyclin, CYCA1;2, also named TARDY ASYNCHRONOUS MEIOSIS (TAM), is known for its positive role in meiotic cell cycle progression, but its function in other cells has not been characterized. This paper reports the role of CYCA1;2/TAM in differentiated cells in vegetative organs. The pattern of CYCA1;2/TAM expression was investigated by promoter and protein fusions using the β-glucuronidase and the green fluorescent protein, respectively. The relevance of the promoter region used in these gene fusion constructs was verified by the effective complementation of the phenotype of the diploid null allele, tam-2 2C by a genomic fragment containing the wild-type coding region of CYCA1;2/TAM and the promoter region. CYCA1;2/TAM expression was found primarily in non-proliferating cells such as guard cells, trichomes, and mesophyll cells, and in vascular tissue. In two types of overexpression lines, one containing the CYCA1;2/TAM transgene driven by the ARABIDOPSIS SKP1-LIKE1 (ASK1) promoter and the other CYCA1;2/TAM-GFP driven by the cauliflower mosaic virus 35S promoter, the largest differences between the transgene transcript levels were approximately 72- and 45-folds, respectively, but the TAM-GFP signal levels in the mesophyll and stomata in the 35S:TAM-GFP lines only differ slightly. Furthermore, the GFP signals in the mesophyll and stomata in the TAM:TAM-GFP and 35S:TAM-GFP lines were all at similarly low levels. These results indicate that the CYCA1;2/TAM protein is likely maintained at low levels in these cells through post-transcriptional regulation. Loss of function in CYCA1;2/TAM resulted in increases in the nuclear size in both trichomes and guard cells. Surprisingly, overexpression of CYCA1;2/TAM led to similar increases. The large increases in trichome nuclear size likely reflected ploidy increases while the moderate increases in guard cell nuclear size did not justify for a ploidy increase. These nuclear size increases were not clearly correlated with trichome branch number increases and guard cell size increases, respectively. These results suggest that cellular homeostasis of the CYCA1;2/TAM protein is linked to the control of nuclear sizes in trichomes and guard cells.

TheArabidopsisF-Box Protein CORONATINE INSENSITIVE1 Is Stabilized by SCFCOI1 and Degraded via the 26S Proteasome Pathway

Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCF(COI1)) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCF(COI1) complexes, suggesting a function for SCF(COI1) in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCF(COI1)-mediated stabilization and 26S proteasome-mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses.

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.

The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein acts predominately from leptotene to pachytene and represses homologous recombination in male meiosis

Normal progression of genetic recombination requires timely degradation of many proteins, but little is known about the proteolytic mechanism. The ARABIDOPSIS SKP1-LIKE1 (ASK1) protein is a component of the Skp1-Cullin-F-box-protein (SCF) ubiquitin ligases that target a variety of proteins for degradation via the 26S proteasome pathway. Previous studies indicate that the early defects of the mutant ask1-1 occur in a prophase-I period overlapping with the period of homologous recombination. We provide evidence in this report that ASK1 is predominately expressed from leptotene to pachytene, and negatively regulates recombination. First, the ASK1 transcript was found not to co-exist with that of its closest homolog ASK2 only during prophase I of male meiosis, suggesting that ASK1 is functionally non-redundant only in prophase I. Second, the peak level of an ASK1-green fluorescence protein (GFP) fusion protein expressed by an ASK1 promoter region occurred only from leptotene to pachytene. The ASK1-GFP in a dominant negative fashion resulted in abnormal tetrads resembling those of the ask1-1 mutant, supporting that the expression timing of the ASK1-GFP in male meiocytes reflects the expression timing of the endogenous ASK1. Lastly, using a marker for recombination events, a significant increase in recombination frequency was detected in plants heterozygous for ask1-1. These results indicate that ASK1 normally plays a repressive role in male recombination in Arabidopsis.

Molecular analyses of the Arabidopsis TUBBY-like protein gene family.

In mammals, TUBBY-like proteins play an important role in maintenance and function of neuronal cells during postdifferentiation and development. We have identified a TUBBY-like protein gene family with 11 members in Arabidopsis, named AtTLP1-11. Although seven of the AtTLP genes are located on chromosome I, no local tandem repeats or gene clusters are identified. Except for AtTLP4, reverse transcription-PCR analysis indicates that all these genes are expressed in various organs in 6-week-old Arabidopsis. AtTLP1, 2, 3, 6, 7, 9, 10, and 11 are expressed ubiquitously in all the organs tested, but the expression of AtTLP5 and 8 shows dramatic organ specificity. These 11 family members share 30% to 80% amino acid similarities across their conserved C-terminal tubby domains. Unlike the highly diverse N-terminal region of animal TUBBY-like proteins, all AtTLP members except AtTLP8 contain a conserved F-box domain (51-57 residues). The interaction between AtTLP9 and ASK1 (Arabidopsis Skp1-like 1) is confirmed via yeast (Saccharomyces cerevisiae) two-hybrid assays. Abscisic acid (ABA)-insensitive phenotypes are observed for two independent AtTLP9 mutant lines, whereas transgenic plants overexpressing AtTLP9 are hypersensitive to ABA. These results suggest that AtTLP9 may participate in the ABA signaling pathway.

Defects in nucleolar migration and synapsis in male prophase�I in the ask1-1 mutant of Arabidopsis

The migration of nucleolus to the nuclear periphery and the onset of homologous chromosome synapsis are characteristics of the leptotene to zygotene transition in meiocytes. Little is known about the genes regulating these processes in eukaryotes. Here we report the characterization of morphological defects in prophase I in microsporocytes in the Arabidopsis skp1-like1 (ask1-1) mutant. In ask1-1, the nucleolus of the microsporocyte failed to move to the nuclear periphery throughout prophase I, and the chromosomes failed to synapse, despite the formation of bivalents in late prophase I. We also found that the transcription of the DMC1 gene and ASY1 gene, which were predominantly transcribed prior to zygotene in microsporocytes, was not affected in ask1-1. These findings, and the fact that ASK1 is a component of the SCF ubiquitin ligase, suggest that ASK1 regulates either the leptotene to zygotene transition or events associated with the developmental transition during male meiosis in Arabidopsis.

ASK1 physically interacts with COI1 and is required for male fertility inArabidopsis

Jasmonates are a new class of plant hormones that play important roles in plant development and plant defense. The COI1 gene was previously shown to be required for jasmonate-regulated plant fertility and defense. We demonstrated for the first time that COI1 interacts with the Arabidopsis SKP1-LIKE1 (ASK1) to form a complex that is required for jasmonate action in planta. Functional analysis by antisense strategy showed that ASK1 is involved in male fertility.

ORE9, an F-box protein that regulates leaf senescence in Arabidopsis.

Senescence is a sequence of biochemical and physiological events that constitute the final stage of development. The identification of genes that alter senescence has practical value and is helpful in revealing pathways that influence senescence. However, the genetic mechanisms of senescence are largely unknown. The leaf of the oresara9 (ore9) mutant of Arabidopsis exhibits increased longevity during age-dependent natural senescence by delaying the onset of various senescence symptoms. It also displays delayed senescence symptoms during hormone-modulated senescence. Map-based cloning of ORE9 identified a 693-amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1-like 1), a component of the plant SCF complex. These results suggest that ORE9 functions to limit leaf longevity by removing, through ubiquitin-dependent proteolysis, target proteins that are required to delay the leaf senescence program in Arabidopsis.

The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation.

The yeast and human SKP1 genes regulate the mitotic cell cycle but are not yet known to be required for meiosis. Nine Arabidopsis SKP1 homologues have been uncovered and are named ASK1 through ASK9. Here, we report the isolation and characterization of a male sterile Arabidopsis mutant and show that the mutant defect was caused by a Ds transposon insertion into the ASK1 gene. In the ask1-1 mutant, abnormal microspores exhibit a range of sizes. Furthermore, during mutant male meiosis, although homologous chromosome pairing appeared normal at metaphase I, chromosome segregation at anaphase I is unequal, and some chromosomes are abnormally extended. Therefore, in ask1-1, at least some homologues remain associated after metaphase I. In addition, immunofluorescence microscopy indicates that the mutant spindle morphology at both metaphase I and early anaphase I is normal; thus, the abnormal chromosome segregation is not likely caused by a spindle defect. Because the yeast Skp1p is required for targeting specific proteins for ubiquitin-mediated proteolysis, we propose that ASK1 controls homologue separation by degrading or otherwise removing a protein that is required directly or indirectly for homologue association before anaphase I.