Genome-Wide Identification and Expression of Xenopus F-Box Family of Proteins

Protein degradation via the multistep ubiquitin/26S proteasome pathway is a rapid way to alter the protein profile and drive cell processes and developmental changes. Many key regulators of embryonic development are targeted for degradation by E3 ubiquitin ligases. The most studied family of E3 ubiquitin ligases is the SCF ubiquitin ligases, which use F-box adaptor proteins to recognize and recruit target proteins. Here, we used a bioinformatics screen and phylogenetic analysis to identify and annotate the family of F-box proteins in the Xenopus tropicalis genome. To shed light on the function of the F-box proteins, we analyzed expression of F-box genes during early stages of Xenopus development. Many F-box genes are broadly expressed with expression domains localized to diverse tissues including brain, spinal cord, eye, neural crest derivatives, somites, kidneys, and heart. All together, our genome-wide identification and expression profiling of the Xenopus F-box family of proteins provide a foundation for future research aimed to identify the precise role of F-box dependent E3 ubiquitin ligases and their targets in the regulatory circuits of development.     

The F-box gene family is expanded in herbaceous annual plants relative to woody perennial plants

F-box proteins are generally responsible for substrate recognition in the Skp1-Cullin-F-box complexes that are involved in protein degradation via the ubiquitin-26S proteasome pathway. In plants, F-box genes influence a variety of biological processes, such as leaf senescence, branching, self-incompatibility, and responses to biotic and abiotic stresses. The number of F-box genes in Populus (Populus trichocarpa; approximately 320) is less than half that found in Arabidopsis (Arabidopsis thaliana; approximately 660) or Oryza (Oryza sativa; approximately 680), even though the total number of genes in Populus is equivalent to that in Oryza and 1.5 times that in Arabidopsis. We performed comparative genomics analysis between the woody perennial plant Populus and the herbaceous annual plants Arabidopsis and Oryza in order to explicate the functional implications of this large gene family. Our analyses reveal interspecific differences in genomic distribution, orthologous relationship, intron evolution, protein domain structure, and gene expression. The set of F-box genes shared by these species appear to be involved in core biological processes essential for plant growth and development; lineage-specific differences primarily occurred because of an expansion of the F-box genes via tandem duplications in Arabidopsis and Oryza. The number of F-box genes in the newly sequenced woody species Vitis (Vitis vinifera; 156) and Carica (Carica papaya; 139) is similar to that in Populus, supporting the hypothesis that the F-box gene family is expanded in herbaceous annual plants relative to woody perennial plants. This study provides insights into the relationship between the structure and composition of the F-box gene family in herbaceous and woody species and their associated developmental and physiological features.     

A combination of the F-box motif and kelch repeats defines a large Arabidopsis family of F-box proteins

In the sequences released by the Arabidopsis Genome Initiative (AGI), we have discovered a new large gene family (48 genes as of July 2000). A detailed computational and biochemical analysis of the predicted gene products reveals a novel family of plant F-box proteins, where the amino (N)-terminal F-box motif is followed by four kelch repeats and a characteristic carboxy-terminal domain. F-box proteins are an expanding family of eukaryotic proteins, which have been shown in some cases to be critical for the controlled degradation of cellular regulatory proteins via the ubiquitin pathway. The F-box motif of the At5g48990 gene product, a member of the family, was shown to be functionally active by its ability to mediate the in vitro interaction between At5g48990 and ASK1 proteins. F-box proteins specifically recruit the targets to be ubiquitinated, mainly through protein-protein interaction modules such as WD-40 domains or leucine-rich repeats (LRRs). The kelch repeats of the family described here form a potential protein-protein interaction domain, as molecular modelling of the kelch repeats according to the galactose oxidase crystal structure (the only solved structure containing kelch repeats) predicts a -propeller. The identification of this family of F-box proteins greatly expands the field of plant F-box proteins and suggests that controlled degradation of cellular proteins via the ubiquitin pathway could play a critical role in multiple plant cellular processes.     

Genome-wide identification and characterisation of F-box family in maize

F-box-containing proteins, as the key components of the protein degradation machinery, are widely distributed in higher plants and are considered as one of the largest known families of regulatory proteins. The F-box protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, systematic analysis of the F-box family in maize (Zea mays) has not been reported yet. In this paper, we identified and characterised the maize F-box genes in a genome-wide scale, including phylogenetic analysis, chromosome distribution, gene structure, promoter analysis and gene expression profiles. A total of 359 F-box genes were identified and divided into 15 subgroups by phylogenetic analysis. The F-box domain was relatively conserved, whereas additional motifs outside the F-box domain may indicate the functional diversification of maize F-box genes. These genes were unevenly distributed in ten maize chromosomes, suggesting that they expanded in the maize genome because of tandem and segmental duplication events. The expression profiles suggested that the maize F-box genes had temporal and spatial expression patterns. Putative cis-acting regulatory DNA elements involved in abiotic stresses were observed in maize F-box gene promoters. The gene expression profiles under abiotic stresses also suggested that some genes participated in stress responsive pathways. Furthermore, ten genes were chosen for quantitative real-time PCR analysis under drought stress and the results were consistent with the microarray data. This study has produced a comparative genomics analysis of the maize ZmFBX gene family that can be used in further studies to uncover their roles in maize growth and development.     

The Involvement of Wheat F-Box Protein Gene TaFBA1 in the Oxidative Stress Tolerance of Plants

As one of the largest gene families, F-box domain proteins have been found to play important roles in abiotic stress responses via the ubiquitin pathway. TaFBA1 encodes a homologous F-box protein contained in E3 ubiquitin ligases. In our previous study, we found that the overexpression of TaFBA1 enhanced drought tolerance in transgenic plants. To investigate the mechanisms involved, in this study, we investigated the tolerance of the transgenic plants to oxidative stress. Methyl viologen was used to induce oxidative stress conditions. Real-time PCR and western blot analysis revealed that TaFBA1 expression was up-regulated by oxidative stress treatments. Under oxidative stress conditions, the transgenic tobacco plants showed a higher germination rate, higher root length and less growth inhibition than wild type (WT). The enhanced oxidative stress tolerance of the transgenic plants was also indicated by lower reactive oxygen species (ROS) accumulation, malondialdehyde (MDA) content and cell membrane damage under oxidative stress compared with WT. Higher activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POD), were observed in the transgenic plants than those in WT, which may be related to the upregulated expression of some antioxidant genes via the overexpression of TaFBA1. In others, some stress responsive elements were found in the promoter region of TaFBA1, and TaFBA1 was located in the nucleus, cytoplasm and plasma membrane. These results suggest that TaFBA1 plays an important role in the oxidative stress tolerance of plants. This is important for understanding the functions of F-box proteins in plants’ tolerance to multiple stress conditions.     

Evolutionary Genomics and Adaptive Evolution of the Hedgehog Gene Family (Shh,Ihh and Dhh) in Vertebrates

The Hedgehog (Hh) gene family codes for a class of secreted proteins composed of two active domains that act as signalling molecules during embryo development, namely for the development of the nervous and skeletal systems and the formation of the testis cord. While only one Hh gene is found typically in invertebrate genomes, most vertebrates species have three (Sonic hedgehog – Shh; Indian hedgehog – Ihh; and Desert hedgehog – Dhh), each with different expression patterns and functions, which likely helped promote the increasing complexity of vertebrates and their successful diversification. In this study, we used comparative genomic and adaptive evolutionary analyses to characterize the evolution of the Hh genes in vertebrates following the two major whole genome duplication (WGD) events. To overcome the lack of Hh-coding sequences on avian publicly available databases, we used an extensive dataset of 45 avian and three non-avian reptilian genomes to show that birds have all three Hh paralogs. We find suggestions that following the WGD events, vertebrate Hh paralogous genes evolved independently within similar linkage groups and under different evolutionary rates, especially within the catalytic domain. The structural regions around the ion-binding site were identified to be under positive selection in the signaling domain. These findings contrast with those observed in invertebrates, where different lineages that experienced gene duplication retained similar selective constraints in the Hh orthologs. Our results provide new insights on the evolutionary history of the Hh gene family, the functional roles of these paralogs in vertebrate species, and on the location of mutational hotspots.     

Genome-Wide Identification of the F-box Gene Family and Expression Analysis under Drought and Salt Stress in Barley

The F-box protein-encoding gene family plays an essential role in plant stress resistance. In present study, 126 non-redundant F-box genes were identified in barley (Hordeum vulgare L., Hv). The corresponding proteins contained 165– 887 amino acid residues and all were amphiphilic, except 5 proteins. Phylogenetic analysis of F-box protein sequences in barley and stress-related F-box protein sequences in wheat and Arabidopsis thaliana (At) was used to classify barley F-box genes are divided into 9 subfamilies (A–I). A structure-based sequence alignment demonstrated that F-box proteins were highly conserved with a total of 10 conserved motifs. In total, 124 F-box genes were unevenly distributed on 7 chromosomes; another 2 genes have not been anchored yet. The gene structure analysis revealed high variability in the number of exons and introns in F-box genes. Comprehensive analysis of expression profiles and phylogenetic tree analysis, a total of 12 F-box genes that may be related to stress tolerance in barley were screened. Of the 12 detected F-box genes, 8 and 10 were upregulated after drought and salt stress treatments, respectively, using quantitative real-time polymerase chain reaction (qRT-PCR). This study is the first systematic analysis conducted on the F-box gene family in barley, which is of great importance for clarifying this family’s bioinformatic characteristics and elucidating its function in barley stress resistance. These results will serve as a theoretical reference for subsequent research on molecular regulation mechanisms, genetic breeding, and improvement.     

Functional Divergence and Evolutionary Dynamics of the Putative AAAP Gene Family in Brassica rapa

The amino acid/auxin permease (AAAP) protein family is ubiquitously present in almost all eukaryotic species and functions in various aspects of growth and development. To investigate the evolution of AAAP proteins, here 83 AAAP genes in Brassica rapa were identified, and their sequence features, and evolutionary relationships were analyzed using in silico methods. According to the phylogenetic analysis, the AAAP genes of B. rapa are divided into six clades, and these clades share relatively similar sequence features, including gene structures, conserved motifs, and domain organizations. Synteny mapping strongly suggested that segmental duplications could be responsible for the expansion of this family. Adaptive evolution analysis demonstrated that most of AAAP proteins were subject to purifying selection. However, the site Tyr²⁵⁷ on eight AAAP proteins from clade 2b underwent significant positive selection. Functional divergent analysis showed that type I functional divergence coefficients (θ _I ) were significantly greater than zero in six pair-wise comparisons. However, functional divergence sites (Q _k ?>?0.95) found only in the AAAP I/II and AAAP I/III comparisons were localized mainly to the trans-membrane (TM) regions, suggesting highly divergent TM structures between these groups might be associated with group-specific functions. Our results could provide a valuable clue for further investigations of the evolutionary history and biological functions of the AAAP genes in B. rapa.     

The IQD Gene Family in Soybean: Structure,Phylogeny, Evolution and Expression

Members of the plant-specific IQ67-domain (IQD) protein family are involved in plant development and the basal defense response. Although systematic characterization of this family has been carried out in Arabidopsis, tomato (Solanum lycopersicum), Brachypodium distachyon and rice (Oryza sativa), systematic analysis and expression profiling of this gene family in soybean (Glycine max) have not previously been reported. In this study, we identified and structurally characterized IQD genes in the soybean genome. A complete set of 67 soybean IQD genes (GmIQD1–67) was identified using Blast search tools, and the genes were clustered into four subfamilies (IQD I–IV) based on phylogeny. These soybean IQD genes are distributed unevenly across all 20 chromosomes, with 30 segmental duplication events, suggesting that segmental duplication has played a major role in the expansion of the soybean IQD gene family. Analysis of the Ka/Ks ratios showed that the duplicated genes of the GmIQD family primarily underwent purifying selection. Microsynteny was detected in most pairs: genes in clade 1–3 might be present in genome regions that were inverted, expanded or contracted after the divergence; most gene pairs in clade 4 showed high conservation with little rearrangement among these gene-residing regions. Of the soybean IQD genes examined, six were most highly expressed in young leaves, six in flowers, one in roots and two in nodules. Our qRT-PCR analysis of 24 soybean IQD III genes confirmed that these genes are regulated by MeJA stress. Our findings present a comprehensive overview of the soybean IQD gene family and provide insights into the evolution of this family. In addition, this work lays a solid foundation for further experiments aimed at determining the biological functions of soybean IQD genes in growth and development.     

Genomewide comparative phylogenetic and molecular evolutionary analysis of tubby-like protein family in Arabidopsis, rice, and poplar

Tubby-like proteins, which are characterized by a highly conserved tubby domain, play an important role in the maintenance and function of neuronal cells during postdifferentiation and development in mammals. In additional to the tubby domain, most tubby-like proteins in plants also possess an F-box domain. Plants also appear to harbor a large number of TLP genes. To gain insight into how TLP genes evolved in plants, we conducted a comparative phylogenetic and molecular evolutionary analysis of the tubby-like protein gene family in Arabidopsis, rice, and poplar. Genomewide screening identified 11 TLP genes in Arabidopsis, 14 in rice, and 11 in poplar. Phylogenetic trees, domain organizations, and intron/exon structures classified this family into three subfamilies and indicated that species-specific expansion contributed to the evolution of this family in plants. We determined that in rice and poplar, the tubby-like protein family had expanded mainly through segmental duplication events. Tissue-specific expression analysis indicated that functional diversification of the duplicated TLP genes was a major feature of long-term evolution. Our results also demonstrated that the tubby and F-box domains had co-evolved during the evolution of proteins containing both domains.     

Characterization of a F-box gene up-regulated by phytohormones and upon biotic and abiotic stresses in grapevine

F-box proteins are key components of the ubiquitin (Ub)/26S proteasome pathway that mediates selective degradation of regulatory proteins involved in a wide variety of cellular processes affecting eukaryotic cells. In plants, F-box genes form one of the largest multigene superfamilies and control many important biological functions. Among the F-box genes characterized to date only few have been involved in the regulation of plant defense responses. Moreover, no F-box genes have been studied and characterized in grapevine. Using a differential display approach we isolated a F-box gene (BIG-24.1), which is up-regulated during Botrytis cinerea infection of grapevine leaves. BIG-24.1 encodes a polypeptide of 386 amino acids with a conserved F-box domain in the N-terminus region and a kelch domain. By investigating expression profiles of BIG-24.1, we show that the gene expression is strongly stimulated in B. cinerea infected berries and in grapevine cells challenged by MAMP rhamnolipids, a non-host bacterium and an endophytic rhizobacterium. The gene is also strongly induced by abiotic stresses including UV-C and wounding or by salicylic acid, methyl-jasmonate, ethylene and abscisic acid that are known to be involved in defense signalling pathways. In addition, sequence analysis of the BIG-24.1 promoter revealed the presence of several regulatory elements involved in the activation of plant defense responses.     

Dissecting the function of Cullin-RING ubiquitin ligase complex genes in planarian regeneration

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, ?3 and ?4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo.     

Molecular evolution and expression of the CRAL_TRIO protein family in insects

CRAL_TRIO domain proteins are known to bind small lipophilic molecules such as retinal, inositol and Vitamin E and include such gene family members as PINTA, α-tocopherol transfer (ATT) proteins, retinoid binding proteins, and clavesins. In insects, very little is known about either the molecular evolution of this family of proteins or their ligand specificity. Here we characterize insect CRAL_TRIO domain proteins and present the first insect CRAL_TRIO protein phylogeny constructed by performing reciprocal BLAST searches of the reference genomes of Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum, Bombyx mori, Manduca sexta and Danaus plexippus. We find several highly conserved amino acid residues in the CRAL_TRIO domain-containing genes across insects and a gene expansion resulting in more than twice as many gene family members in lepidopterans than in other surveyed insect species, but no lepidopteran homolog of the PINTA gene in Drosophila. In addition, we examined the expression pattern of CRAL_TRIO domain genes in Manduca sexta heads using RNA-Seq data. Of the 42 gene family members found in the M. sexta reference genome, we found 30 expressed in the head tissue with similar expression profiles between males and females. Our results suggest this gene family underwent a large expansion in Lepidoptera, making the lepidopteran CRAL_TRIO domain family distinct from other holometabolous insect lineages.     

The intronic region of Fbxl12 functions as an alternative promoter regulated by UV irradiation

The ubiquitin ligases, SCF complexes, consist of Cul1, Skp1, Rbx1 and the substrate recognition components F-box proteins. Previous studies have reported that one of these F-box proteins, Fbl12, which is produced by Fbxl12 gene, regulates both cell cycle and differentiation. In this paper, we show that the intronic region of Fbxl12 gene acts as an alternative promoter and induces expression of a short form of Fbl12 that lacks F-box domain (Fbl12ΔF). We also found that UV irradiation increases Fbl12ΔF mRNA in cells. Finally, Fbl12ΔF may promote the subcellular localization of Fbl12 from nucleus to cytoplasm through their binding. Our data provide the possibility that Fbl12ΔF induced by alternative promoter controls the SCF^Fbl12 activity in response to UV stimulation.