Genome-wide analysis and characterization of F-box gene family in Gossypium hirsutum L.

BACKGROUND: F-box proteins are substrate-recognition components of the Skp1-Rbx1-Cul1-F-box protein (SCF) ubiquitin ligases. By selectively targeting the key regulatory proteins or enzymes for ubiquitination and 26S proteasome mediated degradation, F-box proteins play diverse roles in plant growth/development and in the responses of plants to both environmental and endogenous signals. Studies of F-box proteins from the model plant Arabidopsis and from many additional plant species have demonstrated that they belong to a super gene family, and function across almost all aspects of the plant life cycle. However, systematic exploration of F-box family genes in the important fiber crop cotton (Gossypium hirsutum) has not been previously performed. The genome-wide analysis of the cotton F-box gene family is now possible thanks to the completion of several cotton genome sequencing projects. RESULTS: In current study, we first conducted a genome-wide investigation of cotton F-box family genes by reference to the published F-box protein sequences from other plant species. 592 F-box protein encoding genes were identified in the Gossypium hirsutume acc.TM-1 genome and, subsequently, we were able to present their gene structures, chromosomal locations, syntenic relationships with their parent species. In addition, duplication modes analysis showed that cotton F-box genes were distributed to 26 chromosomes, with the maximum number of genes being detected on chromosome 5. Although the WGD (whole-genome duplication) mode seems play a dominant role during cotton F-box gene expansion process, other duplication modes including TD (tandem duplication), PD (proximal duplication), and TRD (transposed duplication) also contribute significantly to the evolutionary expansion of cotton F-box genes. Collectively, these bioinformatic analysis suggest possible evolutionary forces underlying F-box gene diversification. Additionally, we also conducted analyses of gene ontology, and expression profiles in silico, allowing identification of F-box gene members potentially involved in hormone signal transduction. CONCLUSION: The results of this study provide first insights into the Gossypium hirsutum F-box gene family, which lays the foundation for future studies of functionality, particularly those involving F-box protein family members that play a role in hormone signal transduction.

SCF(KMD) controls cytokinin signaling by regulating the degradation of type-B response regulators.

Cytokinins are plant hormones that play critical roles in growth and development. In Arabidopsis, the transcriptional response to cytokinin is regulated by action of type-B Arabidopsis response regulators (ARRs). Although central elements in the cytokinin signal transduction pathway have been identified, mechanisms controlling output remain to be elucidated. Here we demonstrate that a family of F-box proteins, called the kiss me deadly (KMD) family, targets type-B ARR proteins for degradation. KMD proteins form an S-phase kinase-associated PROTEIN1 (SKP1)/Cullin/F-box protein (SCF) E3 ubiquitin ligase complex and directly interact with type-B ARR proteins. Loss-of-function KMD mutants stabilize type-B ARRs and exhibit an enhanced cytokinin response. In contrast, plants with elevated KMD expression destabilize type-B ARR proteins leading to cytokinin insensitivity. Our results support a model in which an SCF(KMD) complex negatively regulates cytokinin responses by controlling levels of a key family of transcription factors.

Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome.

The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40-50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of the Skp1-cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes (F-box genes for short) were identified and further subdivided into 12 groups according to the characterization of known functional domains, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain. The F-box genes were distributed in all 17 apple chromosomes with various densities and tended to form gene clusters. Spatial expression profile analysis revealed that F-box genes have organ-specific expression and are widely expressed in all organs. Proteins that contained the galactose oxidase domain were highly expressed in leaves, flowers and seeds. From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. The results of this study helped to elucidate the functions of F-box proteins, especially in Rosaceae plants.

An anther development F-box (ADF) protein regulated by tapetum degeneration retardation (TDR) controls rice anther development.

MAIN CONCLUSION: In this study, we reported that a F-box protein, OsADF, as one of the direct targets of TDR , plays a critical role in rice tapetum cell development and pollen formation. The tapetum, the innermost sporophytic tissue of anther, plays an important supportive role in male reproduction in flowering plants. After meiosis, tapetal cells undergo programmed cell death (PCD) and provide nutrients for pollen development. Previously we showed that tapetum degeneration retardation (TDR), a basic helix-loop-helix transcription factor, can trigger tapetal PCD and control pollen wall development during anther development. However, the comprehensive regulatory network of TDR remains to be investigated. In this study, we cloned and characterized a panicle-specific expression F-box protein, anther development F-box (OsADF). By qRT-PCR and RNA in situ hybridization, we further confirmed that OsADF expressed specially in tapetal cells from stage 9 to stage 12 during anther development. In consistent with this specific expression pattern, the RNAi transgenic lines of OsADF exhibited abnormal tapetal degeneration and aborted microspores development, which eventually grew pollens with reduced fertility. Furthermore, we demonstrated that the TDR, a key regulator in controlling rice anther development, could regulate directly the expression of OsADF by binding to E-box motifs of its promoter. Therefore, this work highlighted the possible regulatory role of TDR, which regulates tapetal cell development and pollen formation via triggering the possible ADF-mediated proteolysis pathway.

Evolution of F-box genes in plants: different modes of sequence divergence and their relationships with functional diversification.

F-box proteins are substrate-recognition components of the Skp1-Rbx1-Cul1-F-box protein (SCF) ubiquitin ligases. In plants, F-box genes form one of the largest multigene superfamilies and control many important biological functions. However, it is unclear how and why plants have acquired a large number of F-box genes. Here we identified 692, 337, and 779 F-box genes in Arabidopsis, poplar and rice, respectively, and studied their phylogenetic relationships and evolutionary patterns. We found that the plant F-box superfamily can be divided into 42 families, each of which has a distinct domain organization. We also estimated the number of ancestral genes for each family and identified highly conservative versus divergent families. In conservative families, there has been little or no change in the number of genes since the divergence between eudicots and monocots approximately 145 million years ago. In divergent families, however, the numbers have increased dramatically during the same period. In two cases, the numbers of genes in extant species are >100 times greater than that in the most recent common ancestor (MRCA) of the three species. Proteins encoded by highly conservative genes always have the same domain organization, suggesting that they interact with the same or similar substrates. In contrast, proteins of rapidly duplicating genes sometimes have quite different domain structures, mainly caused by unusually frequent shifts of exon-intron boundaries and/or frameshift mutations. Our results indicate that different F-box families, or different clusters of the same family, have experienced dramatically different modes of sequence divergence, apparently having resulted in adaptive changes in function.

Genome-wide characterization of the hyperaccumulator Sedum alfredii F-box family under cadmium stress.

The F-box genes, which form one of the largest gene families in plants, are vital for plant growth, development and stress response. However, F-box gene family in Sedum alfredii remains unknown. Comprehensive studies addressing their function responding to cadmium stress is still limited. In the present study, 193 members of the F-box gene (SaFbox) family were identified, which were classified into nine subfamilies. Most of the SaFboxs had highly conserved domain and motif. Various functionally related cis-elements involved in plant growth regulation, stress and hormone responses were located in the upstream regions of SaFbox genes. RNA-sequencing and co-expression network analysis revealed that the identified SaFbox genes would be involved in Cd stress. Expression analysis of 16 hub genes confirmed their transcription level in different tissues. Four hub genes (SaFbox40, SaFbox51, SaFbox136 and SaFbox170) were heterologously expressed in a Cd-sensitive yeast cell to assess their effects on Cd tolerance. The transgenic yeast cells carrying SaFbox40, SaFbox51, SaFbox136, or SaFbox170 were more sensitive and accumulated more cadmium under Cd stress than empty vector transformed control cells. Our results performed a comprehensive analysis of Fboxs in S. alfredii and identified their potential roles in Cd stress response.

Genome-wide survey of the F-box/Kelch (FBK) members and molecular identification of a novel FBK gene TaAFR in wheat.

F-box proteins play critical roles in plant responses to biotic/abiotic stresses. In the present study, a total of 68 wheat F-box/Kelch (TaFBK) genes, unevenly distributed across 21 chromosomes and encoding 74 proteins, were identified in EnsemblPlants. Protein sequences were compared with those of Arabidopsis and three cereal species by phylogenetic and domain analyses, where the wheat sequences were resolved into 6 clades. In silico analysis of a digital PCR dataset revealed that TaFBKs were expressed at multiple developmental stages and tissues, and in response to drought and/or heat stresses. The TaFBK19 gene, a homolog of the Attenuated Far-Red Response (AFR) genes in other plant species, and hence named TaAFR, was selected for further analysis. Reverse-transcription quantitative real-time PCR (RT-qPCR) was carried out to determine tissue-specific, hormone and stress (abiotic/biotic) responsive expression patterns. Of interest, TaAFR was expressed most abundantly in the leaves, and its expression in response to leaf rust variants suggests a potential role in compatible vs incompatible rust responses. The protein was predicted to localize in cytosol, but it was shown experimentally to localize in both the cytosol and the nucleus of tobacco. A series of protein interaction studies, starting with a yeast-2-hybrid (Y2H) library screen (wheat leaf infected with incompatible leaf rust pathogens), led to the identification of three TaAFR interacting proteins. Skp1/ASK1-like protein (Skp1) was found to interact with the F-box domain of TaAFR, while ADP-ribosylation factor 2-like isoform X1 (ARL2) and phenylalanine ammonia-lyase (PAL) were shown to interact with its Kelch domain. The data presented herein provides a solid foundation from which the function and metabolic network of TaAFR and other wheat FBKs can be further explored.

Genetic features of the spontaneous self-compatible mutant, 'Jin Zhui' (Pyrus bretschneideri Rehd.).

'Jin Zhui' is a spontaneous self-compatible mutant of 'Ya Li' (Pyrus bretschneideri Rehd. S21S34 ), the latter displaying a typical S-RNase-based gametophytic self-incompatibility (GSI). The pollen-part mutation (PPM) of 'Jin Zhui' might be due to a natural mutation in the pollen-S gene (S34 haplotype). However, the molecular mechanisms behind these phenotypic changes are still unclear. In this study, we identified five SLF (S-Locus F-box) genes in 'Ya Li', while no nucleotide differences were found in the SLF genes of 'Jin Zhui'. Further genetic analysis by S-RNase PCR-typing of selfed progeny of 'Jin Zhui' and 'Ya Li' × 'Jin Zhui' progeny showed three progeny classes (S21S21 , S21S34 and S34S34 ) as opposed to the two classes reported previously (S21S34 and S34S34 ), indicating that the pollen gametes of 'Jin Zhui', bearing either the S21 - or S34 -haplotype, were able to overcome self-incompatibility (SI) barriers. Moreover, no evidence of pollen-S duplication was found. These findings support the hypothesis that loss of function of S-locus unlinked PPM expressed in pollen leads to SI breakdown in 'Jin Zhui', rather than natural mutation in the pollen-S gene (S34 haplotype). Furthermore, abnormal meiosis was observed in a number of pollen mother cells (PMCs) in 'Jin Zhui', but not in 'Ya Li'. These and other interesting findings are discussed.

Diversity in tissue expression, substrate binding, and SCF complex formation for a lectin family of ubiquitin ligases.

Post-translational modification of proteins regulates many cellular processes. Some modifications, including N-linked glycosylation, serve multiple functions. For example, the attachment of N-linked glycans to nascent proteins in the endoplasmic reticulum facilitates proper folding, whereas retention of high mannose glycans on misfolded glycoproteins serves as a signal for retrotranslocation and ubiquitin-mediated proteasomal degradation. Here we examine the substrate specificity of the only family of ubiquitin ligase subunits thought to target glycoproteins through their attached glycans. The five proteins comprising this FBA family (FBXO2, FBXO6, FBXO17, FBXO27, and FBXO44) contain a conserved G domain that mediates substrate binding. Using a variety of complementary approaches, including glycan arrays, we show that each family member has differing specificity for glycosylated substrates. Collectively, the F-box proteins in the FBA family bind high mannose and sulfated glycoproteins, with one FBA protein, FBX044, failing to bind any glycans on the tested arrays. Site-directed mutagenesis of two aromatic amino acids in the G domain demonstrated that the hydrophobic pocket created by these amino acids is necessary for high affinity glycan binding. All FBA proteins co-precipitated components of the canonical SCF complex (Skp1, Cullin1, and Rbx1), yet FBXO2 bound very little Cullin1, suggesting that FBXO2 may exist primarily as a heterodimer with Skp1. Using subunit-specific antibodies, we further demonstrate marked divergence in tissue distribution and developmental expression. These differences in substrate recognition, SCF complex formation, and tissue distribution suggest that FBA proteins play diverse roles in glycoprotein quality control.

F-box proteins in rice. Genome-wide analysis, classification, temporal and spatial gene expression during panicle and seed development, and regulation by light and abiotic stress.

F-box proteins constitute a large family in eukaryotes and are characterized by a conserved F-box motif (approximately 40 amino acids). As components of the Skp1p-cullin-F-box complex, F-box proteins are critical for the controlled degradation of cellular proteins. We have identified 687 potential F-box proteins in rice (Oryza sativa), the model monocotyledonous plant, by a reiterative database search. Computational analysis revealed the presence of several other functional domains, including leucine-rich repeats, kelch repeats, F-box associated domain, domain of unknown function, and tubby domain in F-box proteins. Based upon their domain composition, they have been classified into 10 subfamilies. Several putative novel conserved motifs have been identified in F-box proteins, which do not contain any other known functional domain. An analysis of a complete set of F-box proteins in rice is presented, including classification, chromosomal location, conserved motifs, and phylogenetic relationship. It appears that the expansion of F-box family in rice, in large part, might have occurred due to localized gene duplications. Furthermore, comprehensive digital expression analysis of F-box protein-encoding genes has been complemented with microarray analysis. The results reveal specific and/or overlapping expression of rice F-box protein-encoding genes during floral transition as well as panicle and seed development. At least 43 F-box protein-encoding genes have been found to be differentially expressed in rice seedlings subjected to different abiotic stress conditions. The expression of several F-box protein-encoding genes is also influenced by light. The structure and function of F-box proteins in plants is discussed in light of these results and the published information. These data will be useful for prioritization of F-box proteins for functional validation in rice.

The Arabidopsis EIN3 binding F-Box proteins EBF1 and EBF2 have distinct but overlapping roles in ethylene signaling.

Ethylene signaling in Arabidopsis thaliana converges on the ETHYLENE-INSENSITIVE3 (EIN3)/EIN3-Like (EIL) transcription factors to induce various responses. EIN3 BINDING F-BOX1 (EBF1) and EBF2 were recently shown to function in ethylene perception by regulating EIN3/EIL turnover. In the absence of ethylene, EIN3 and possibly other EIL proteins are targeted for ubiquitination and subsequent degradation by Cullin 1-based E3 complexes containing EBF1 and 2. Ethylene appears to block this ubiquitination, allowing EIN3/EIL levels to rise and mediate ethylene signaling. Through analysis of mutant combinations affecting accumulation of EBF1, EBF2, EIN3, and EIL1, we show that EIN3 and EIL1 are the main targets of EBF1/2. Kinetic analyses of hypocotyl growth inhibition in response to ethylene and growth recovery after removal of the hormone revealed that EBF1 and 2 have temporally distinct but overlapping roles in modulating ethylene perception. Whereas EBF1 plays the main role in air and during the initial phase of signaling, EBF2 plays a more prominent role during the latter stages of the response and the resumption of growth following ethylene removal. Through their coordinated control of EIN3/EIL1 levels, EBF1 and EBF2 fine-tune ethylene responses by repressing signaling in the absence of the hormone, dampening signaling at high hormone concentrations, and promoting a more rapid recovery after ethylene levels dissipate.

Ubiquitylation of cyclin E requires the sequential function of SCF complexes containing distinct hCdc4 isoforms.

Cyclin E, an activator of cyclin-dependent kinase 2 (Cdk2), is targeted for proteasomal degradation by phosphorylation-dependent multiubiquitylation via the ubiquitin ligase SCF(hCdc4). SCF ubiquitin ligases are composed of a core of conserved subunits and one variable subunit (an F box protein) involved in substrate recognition. We show here that multiubiquitylation of cyclin E requires the sequential function of two distinct splice variant isoforms of the F box protein hCdc4 known as alpha and gamma. SCF(hCdc4alpha) binds a complex containing cyclin E, Cdk2, and the prolyl cis/trans isomerase Pin1 and promotes the activity of Pin1 without directly ubiquitylating cyclin E. However, due to the action of this SCF(hCdc4alpha)-Pin1 complex, cyclin E becomes an efficient ubiquitylation substrate of SCF(hCdc4gamma). Furthermore, in the context of Cdc4alpha and cyclin E, mutational data suggest that Pin1 isomerizes a noncanonical proline-proline bond, with the possibility that Cdc4alpha may serve as a cofactor for altering the specificity of Pin1.

The SCF ubiquitin ligase: insights into a molecular machine.

Ubiquitin ligases are well suited to regulate molecular networks that operate on a post-translational timescale. The F-box family of proteins - which are the substrate-recognition components of the Skp1-Cul1-F-box-protein (SCF) ubiquitin ligase - are important players in many mammalian functions. Here we explore a unifying and structurally detailed view of SCF-mediated proteolytic control of cellular processes that has been revealed by recent studies.