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Genome-Wide Characterization and Analysis of F-Box Protein-Encoding

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Genome-Wide Characterization and Analysis of F-Box Protein-Encoding Mol Genet Genomics (2015) 290:1435–1446 DOI 10.1007/s00438-015-1004-z ORIGINAL PAPER Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome Hao-Ran Cui · Zheng-Rong Zhang · Wei lv · Jia-Ning Xu · Xiao-Yun Wang Received: 24 October 2014 / Accepted: 29 January 2015 / Published online: 18 February 2015 © Springer-Verlag Berlin Heidelberg 2015 Abstract The F-box protein family is a large family that Using qRT-PCR to examine the expression of F-box genes is characterized by conserved F-box domains of approxi- encoding proteins with domains related to stress, the results mately 40–50 amino acids in the N-terminus. F-box pro- revealed that F-box proteins were up- or down-regulated, teins participate in diverse cellular processes, such as which suggests that F-box genes were involved in abiotic development of floral organs, signal transduction and stress. The results of this study helped to elucidate the response to stress, primarily as a component of the Skp1- functions of F-box proteins, especially in Rosaceae plants. cullin-F-box (SCF) complex. In this study, using a global search of the apple genome, 517 F-box protein-encoding Keywords Apple · F-box · Bioinformatics · Expression genes (F-box genes for short) were identified and further pattern · Stress 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. Introduction Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was The F-box motif is a conserved sequence that is found present with or without the Kelch domain. The F-box genes widely in proteins from animals and plants. This sequence were distributed in all 17 apple chromosomes with various consists of approximately 40–50 amino acids, which usu- densities and tended to form gene clusters. Spatial expres- ally occur in the N-terminal end of the proteins. Proteins sion profile analysis revealed that F-box genes have organ- with one or more F-box motifs are called F-box proteins. In specific expression and are widely expressed in all organs. animal species, the quantities of predicted F-box protein- Proteins that contained the galactose oxidase domain were encoding genes (F-box genes for short) were generally highly expressed in leaves, flowers and seeds. From a fruit less than a hundred; for example, Drosophila, mice and ripening expression profile, 166 F-box genes were identi- humans had only 27, 74 and 69 F-box genes, respectively fied. The expressions of most of these genes changed little (Kipreos and Pagano 2000; Jin et al. 2004; Jia et al. 2013). during maturation, but five of them increased significantly. However, many more F-box genes were predicted in plant species, for example, 694 F-box genes in Arabidopsis and 687 and 359 in rice and maize, respectively (Gagne et al. Communicated by S. Hohmann. 2002; Jain et al. 2007; Jia et al. 2013). Large numbers of F-box genes implied crucial roles and diverse functions of Electronic supplementary material The online version of this the F-box genes, especially in the plant kingdom. In plants, article (doi:10.1007/s00438-015-1004-z) contains supplementary material, which is available to authorized users. F-box proteins bind with other proteins such as cullin1 and Skp1 to form an SCF complex (Skp1-cullin-F-box com- H.‑R. Cui · Z.‑R. Zhang · W. lv · J.‑N. Xu · X.‑Y. Wang (*) plex), participating in the ubiquitin/26S proteome pathway. College of Life Science, State Key Laboratory of Crop Biology, The ubiquitin/26S proteome pathway is a major posttran- Shandong Agricultural University, Taian 271018, Shandong, People’s Republic of China scriptional regulatory process that allows cells to degrade e-mail: [email protected] redundant proteins through a reaction cascade that requires 1 3 1436 Mol Genet Genomics (2015) 290:1435–1446 three enzymes, an E1 (ubiquitin-activating enzyme), an in the pollen determinants of self-incompatibility (Okada E2 (ubiquitin conjugating enzyme), and an E3 (ubiquitin et al. 2013). The expression of the apple protein TLP7 can ligases). Once ubiquitin is activated by E1, it is transferred enhance stress tolerance in E. coli cells (Du et al. 2014). to E3 under the catalysis of E2 and binds to substrate pro- However, genome-wide analysis of apple F-box genes is teins through E3 (Ciechanover 1998). The SCF complex still yet to be studied. is one of the most well-known E3 s, in which the cullin1, The completion of the apple (Malus domestica) genome Rbx1, and Skp1 subunits comprise the core ligase activity, map offers the possibility of investigating the F-box gene while F-box proteins interact with Skp1 proteins via the family in this species (Velasco et al. 2010). In this study, we F-box domain and bind to substrate proteins through vari- performed a genome-wide search of F-box protein-encod- able protein interaction domains (Xu et al. 2002). ing genes in the apple genome and analyzed their chromo- Since the initial discovery of the F-box protein, cyclin F, somal distributions, functional domains and expression pat- in humans, numerous F-box proteins have been identified terns in different organs and processes. A special domain in eukaryotes. In plants, many F-box proteins are involved called galactose oxidase was first noticed as a functional in a variety of different functions, including hormone sign- domain in F-box proteins and was designated as a distinct aling, defense response, the development of floral organs group in classification. Transcription profile analysis dem- and flowering control, photomorphogenesis, circadian onstrated that F-box genes were expressed in all organs, rhythm, and self-incompatibility. The protein UFO (Unu- and individual F-box genes showed organ-specific expres- sual Floral Organs) is the first identified F-box protein in sion, which suggests that they have different functions. We plants, and it plays roles in the floral meristem and floral also discovered several F-box genes that respond to chill- organ development (Levin and Meyerowitz 1995; Chae ing and fruit ripening. To the best of our knowledge, the et al. 2008). Another set of F-box proteins, including FKF1, present report is the first genome-wide report of the apple ZTL/LKP1 and LKP2, has been found to be involved in F-box family, and our results could provide useful informa- the regulation of the circadian clock, flowering time, and tion for further studies of the apple F-box gene family. photomorphogenesis in Arabidopsis (Kiyosue and Wada 2000; Mas et al. 2003; Somers et al. 2004; Baudry et al. 2010; Takase et al. 2011). A nuclear F-box protein, EID1 Materials and methods (Empfindlicher im Dunkelroten Licht 1), which contains a leucine zipper motif, functions as a negative regulator in Identification and classification of F-box genes in apple phytochrome A-specific light signaling by targeting phy- tochrome A for ubiquitin-dependent proteolysis (Marrocco The genome and proteome sequences of Arabidop- et al. 2006). The F-box protein MAX2 is involved in pho- sis, maize and rice were downloaded from the NCBI ftp tomorphogenesis, including inflorescence architecture and website. Two strategies were used. For the first method, senescence, as a positive regulator. In addition, many F-box BLASTp was performed by the Bioedit program with an proteins have been demonstrated to take part in hormone E value cutoff of 0.001 to search the apple proteome using perception and signaling processes. Several F-box proteins F-box proteins in maize and rice as queries. For the sec- have been found to function as auxin receptors, such as ond method, Hmmer3.0 software was downloaded from the TIR1, AFB1, AFB2 and AFB3, while the F-box proteins HMMER website (http://hmmer.janelia.org/) to perform a AtGID2 and AtSLEEEPY1 are involved in gibberellin sign- global search of the apple proteome. The Hidden Markov aling by directing the SCF complex (McGinnis et al. 2003; Model (HMM) profile of the F-box domain that was used Dill et al. 2004; Dharmasiri et al. 2005; Kepinski and Ley- in this investigation was downloaded from the pfam web- ser 2005; Ariizumi et al. 2011). The F-box proteins EBF1 site (http://pfam.xfam.org/). All of the proteins that were and EBF2 are related to the regulation of plant ethylene obtained by the two strategies were submitted to the Inter- hormone signaling (Potuschak et al. 2003). Another F-box pro Database to ensure the presence of F-box domains. protein, COI1, functions as a receptor for jasmonic acid by Gene sequences were obtained using their corresponding assembling SCF complexes (Thines et al. 2007; Yan et al. numbers in the apple genome with a Bioperl script (http:// 2009). www.rosaceae.org/species/malus/malus_x_domestica/ In apple (Malus domestica), only a few F-box protein genome_v1.0p). The F-box protein sequences that were functions have been discovered. Two homologous apple obtained above were further identified by their conserved F-box proteins, EBF1 and FBCP1, are likely to partici- functional domains through submitting them to the Interpro pate in the ethylene signaling that is involved in the fruit Database for identification, and they were manually clas- ripening process (Han et al. 2008; Tacken et al. 2012). sified into different groups according to their functional F-box proteins in apple have also been found to participate domains. 1 3 Mol Genet Genomics (2015) 290:1435–1446 1437 Sequence logo generation and sequence alignment collected and also stored at 70 °C. The total RNA was − isolated from the leaves using the CTAB procedure (Gasic The apple F-box domain sequences were submitted to et al.
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