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Article

Genome-Wide Identification and Characterization of the OFP Gene Family in the Wild Strawberry Fragaria vesca

by
Xiao Xu
,
Xinyu Wang
,
Sirui Zhou
,
Xumo Huang
,
Pengcheng Liu
,
Bojun Ma
and
Xifeng Chen
*
College of Life Sciences, Zhejiang Normal University, Jinhua 321004, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(3), 569; https://doi.org/10.3390/agronomy14030569
Submission received: 5 February 2024 / Revised: 4 March 2024 / Accepted: 11 March 2024 / Published: 12 March 2024
(This article belongs to the Special Issue Recent Advances in Bioinformatics for Plant Genetic Traits)
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Abstract

:
OVATE family proteins (OFPs) are a kind of plant-specific transcription factor, which play important roles in the growth and development of plants. Here, we performed a genome-wide investigation of the OFP gene family members in the wild diploid strawberry (Fragaria vesca, 2n = 14), and analyzed their physical and chemical properties, gene structure, phylogeny, expression patterns, and the subcellular localizations of these genes. Fourteen OFP genes from F.vesca were identified. Collinearity analysis showed ten pairs of collinearity between F. vesca and Arabidopsis. Phylogenetic analysis divided FvOFP genes into five different clades. The expression patterns of the FvOFP genes assayed in different tissues of F. vesca by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) showed that FvOFP1, FvOFP11, FvOFP12, and FvOFP14 were highly expressed in achene and their expression was further verified in the fruits at different developmental stages. Additionally, the subcellular localizations of FvOFP1, FvOFP11, FvOFP12, and FvOFP14 were preliminarily analyzed in tobacco leaves. The results showed clear fluorescent signals in the nucleus. Our results provided a comprehensive understanding of the potential function of FvOFP genes in strawberries.

1. Introduction

The development of an organism is based on the temporal and spatial regulation of gene expression, in which transcription factors (TFs) act as switches for regulatory cascades [1]. For instance, TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL FACTOR (TCP), play a variety of roles in plant reproductive development, regulating flowering time, the growth of inflorescence stems, and the correct growth and development of flower organs. In Arabidopsis (Arabidopsis thaliana), TCP4 controls leaf size by limiting cell division activity [2]. 3-amino acid loop extension homeodomain (TALE) KNOTTED1-like homeobox (KNOX) transcription factors (TFs), were originally found in maize and play an important role in plant growth and development by regulating the activities of meristems and affecting leaf development [3]. The KNOX mutant kn1, caused by the insertion or tandem repeat of transposable elements, can cause abnormal striations in maize leaves [4]. Another TF, BEL1-like homeodomain (BELL), interacts with KNOX proteins to form a complex network structure. The structure regulates different downstream genes that affect plant development, such as embryo sac development [3].
Recently, the OVATE family proteins (OFPs), forming a plant-specific TF family, were found to control multiple aspects of plant growth and development [5,6]. The OFP genes were first identified in tomato and named OVATE, and regulated fruit shape from oblate to oval [7]. To date, OFP genes have been proven to be widely distributed in the plant kingdom, such as in Arabidopsis (A. thaliana) [6], tomato (Solanum lycopersicum) [8], rice (Oryza sativa) [9], grape (Vitis vinifera) [10], apple (Malus domestica) [11], peach (Prunus persica) [12], radish (Raphanus sativus) [13], cabbage (Brassica rapa ssp. pekinensis) [14], and pepper (Capsicum annuum) [15]. OFPs usually have an approximately 70-amino acid OVATE domain (or named the DUF623 domain) at the C-terminus, which is highly conserved in tomato, rice, and Arabidopsis. OFPs also contain a putative bipartite nuclear localization signal (NLS), and two putative Von Willebrand factor type C (VWFC) domains required for protein–protein interaction, that distinguish OVATEs from any of the previously identified plant genetic regulators [5,7].
Mounting evidence has shown that OFPs play a key role in affecting plant morphological phenotype and suppressing cell elongation by acting as an active transcriptional repressor. The Arabidopsis overexpressing lines of OFP1 exhibited a phenotype of reduced length in all aerial organs, including the hypocotyl, rosette leaf, cauline leaf, inflorescence stem, floral organs, and silique [16,17]. Mutation of OFP5 leads to abnormal egg cells in Arabidopsis [18]. In tomato, a single mutation leading to a premature stop codon, causes the transition of the tomato fruit from round to pear-shaped [7,19,20]. In rice, over-expressing OFP2 reduces plant height and alters leaf morphology, seed shape and the positioning of vascular bundles in the stems [21]. The knockdown of OFP6 results in a semi-dwarf stature, altered grain shape, and shorter lateral roots in rice [22]. OsOFP14 and OsOFP8 have been found to interact with GS9 to regulate grain shape, and knockout of GS9 results in slender grains [23]. In pepper, the suppression of OFPs changes the fruit to be more oblong [15]. Moreover, the morphological regulation mediated by OFPs has been reported to be partly linked with various plant hormones including gibberellic acid (GA), auxin (IAA), salicylic acid (SA), jasmonic acid (JA), and ethylene. OFP1 inhibits GA biosynthesis by suppressing GA20ox1 activity, producing dwarf, rosette-like leaves in Arabidopsis [6]. OsOFP6 affects polar IAA transport to regulate lateral root growth and initiation [22]. The expression of OFP1 was suppressed by ethylene and involved in regulating fruit ripening in banana (Musa acuminata) [24]. In pepper, OFP16, and OFP17 were upregulated by SA, JA, and GA [15]. Additionally, evidence has shown that OFPs also participate in stress response. Overexpression of OFP6 enhanced drought and cold resistance in rice [22]. In apple, the expression level of OFP4 was upregulated under salt and cold stress [11]. In pepper, OFP16 was upregulated under salt and heat stress, while OFP17 was upregulated under cold stress [15]. These facts indicated the potential of OFPs as excellent candidates for modifying complex traits in crops.
Strawberry is one of the most popular fruit crops in the world and is widely enjoyed by consumers for its exquisite appearance and sweet taste. Modern cultivated strawberry (Fragaria × ananassa) is an allo-octoploid (2n = 8x = 56). However, the wild strawberry is diploid, and the genome of the diploid progenitor species Fragaria vesca (2n = 14) has been comprehensively sequenced with high-quality annotations [25,26], thus it has been used as a diploid reference genome for strawberry. Recently, the genome of F. × ananassa with high-quality annotation has also been published [27,28], which will serve as a powerful resource for bioinformatics analysis of gene families in strawberry. As OFPs are proposed to encode a novel TF class and regulate plant development broadly, especially fruit development, further genetic and biochemical studies of the function of these genes in food plants are needed to provide better comprehension of their roles in plant evolution and domestication [9]. Though some OFP genes have been reported in monocotyledonous and dicotyledonous plants, their functions in strawberry are largely unknown. In this study, OFP gene family members were identified in the wild strawberry F. vesca. The basic physical chemistry properties, gene structure, conserved domains, and evolutionary relationships have been comprehensively analyzed. The tissue-specific expression profile and subcellular localization were further characterized. Our results provided a useful reference for future functional analysis of the OFP gene family in strawberry.

2. Materials and Methods

2.1. Plant Material and Growth Condition

The seeds of the F. vesca variety ‘Rugen’ were sown and planted in a greenhouse under 21–23 °C (8 h dark/16 h light) and 70–80% humidity conditions. Seventy days after sowing, tissues including roots, stems, leaves, petals, anthers, and styles were sampled according to the method of Topcu et al. [29]. The strawberry fruits at different growth stages, including green, white, turning, and red were sampled and dissected into two parts (receptacle and achene). These tissues of the plants were collected, respectively, for the analysis of FvOFP gene expression patterns. All the tissues were immediately frozen in liquid nitrogen and stored at −80 °C.

2.2. Identification of OFP Gene Family Members in F. vesca

The amino acid sequences of Arabidopsis AtOFPs (Supplementary Data S1) were used to search for the orthologs from F. vesca with the BLAST tool in the GDR database (https://www.rosaceae.org/, accessed on 1 October 2022) and using the latest F. vesca genome annotation (v4.0.a2). The putative protein sequences of candidate FvOFP genes were further checked for the OFP domain with SMART (http://smart.embl-heidelberg.de/, accessed on 1 October 2022) and NCBI-CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 1 October 2022). The protein sequences of FvOFPs were used to identify the orthologous genes in F. × ananassa (v1.0.a2) from GDR database.

2.3. Analysis of Gene Sequence Characters

Gene annotation gff3 files of F. vesca (v4.0.a2) were downloaded from the GDR database, and the chromosome locations and gene structures of FvOFPs were visualized using TBtools [30]. The motifs of FvOFPs were predicted by MEME (https://meme-suite.org/meme/, accessed on 1 October 2022). Sequence alignment of the OFPs was performed by Mega v7.0. The tertiary structures of the conserved domains with intensive modelling mode were predicted by Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index, accessed on 1 October 2022).

2.4. Syntenic and Phylogenetic Analysis

The synteny of OFP genes between Arabidopsis and F. vesca was analyzed by MCScanX, based on the genomes of these two species and visualized using TBtools v2.042 to show the collinear gene pairs [30,31]. For the phylogeny of OFP genes in plants, the protein sequences of OFP families from F. vesca, A. thaliana [6], S. lycopersicum [8], O. sativa [9], and C. annuum [15] were used, and their protein sequences and gene accessions (https://bioinformatics.psb.ugent.be/plaza/, accessed on 1 October 2022) are shown in Supplementary Data S1. Mega v7.0 was used to construct the neighbour-joining (NJ) phylogenetic trees evaluated by a 1000 replicates bootstrap.

2.5. Gene Expression Assay of the RNA-Seq Data

Orthologs of each FvOFP gene in octoploid F. × ananassa were identified using the genome annotation v1.0.a2 [27]. The F. × ananassa genome-wide RNA-Seq data submitted by Liu et al. [27] were used to dissect the expression pattern of OFP genes at different developmental stages, classified as green, white, turning, and red, in both the receptacle and achene. Transcript per million (TPM) reads values of genes were extracted and visualized as a heatmap using TBtools v2.042 [30].

2.6. Gene Expression Analysis by Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)

Total RNA was extracted using the EASYspin Plus plant RNA kit (AidLab, Beijing, China) and synthesized to the first-strand cDNA using Prime Script RT reagent Kit (TaKaRa, Beijing, China) following the manufacturer’s instructions. TB Green Premix Ex TaqTM (TaKaRa, Beijing, China) was used for qPCR. The thermal cycle was performed on the Step One Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) following: 95 °C for 5 min; 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. The housekeeping gene of F. vesca, FvActin (FvH4_1g03980), was used as the standardization control and the relative expression level of each gene was analyzed using the delta–delta Ct method [32]. Standard errors were calculated based on a minimum of three replicates. The PCR primers are listed in Supplementary Data S2.

2.7. Subcellular Localizations of FvOFPs

Subcellular localizations of FvOFPs were predicted by CELL-PLoc (v.2.0, http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 1 October 2022). The subcellular localizations of FvOFPs were further confirmed by transient expression in tobacco leaf cells. The coding sequence (CDS) of FvOFP genes without the termination codon were amplified from the cDNA of F. vesca by PCR using PrimeSTAR Max DNA Polymerase (TaKaRa, Beijing, China), and fused with the Green fluorescent protein (GFP) gene driven by the CaMV35S promoter in pCAMBIA1300 vector using an In-fusion HD Cloning Kit (TaKaRa, Beijing, China). The constructed vectors were transformed into 4-week-old tobacco (Nicotiana benthamiana) mediated by Agrobacterium (Agrobacterium tumefaciens GV3101), following the method of Bleckmann [33]. After injection, the tobacco plants were cultured at 25 °C under dark treatment for 10 h, then cultured in a growth chamber at 25 °C (16 h light/8 h dark) for 2–3 days. Transformed tobacco leaf cells were observed using a Zeiss LSM880 laser scanning confocal microscope (Zeiss, Oberkochen, Germany) with a 488 nm argon laser.

3. Results

3.1. Genome-Wide Identification of the OPF Gene Family in F. vesca

Using the queries of Arabidopsis AtOFPs with BLAST in the GDR database, a total of 14 putative OFP genes were identified from the wild diploid strawberry F. vesca. These genes were designated as FvOFP1 to FvOFP14 based on their ordered chromosomal positions (Table 1). The alignment of amino acid sequences indicated that the OVATE domain is mostly present at the C-terminus of the FvOFPs (Supplementary Data S3). The CDS and proteins of the FvOFP gene family members are shown in Supplementary Data S4 and S5, respectively. The sequence characters of the FvOFP genes were further characterized. The CDSs of FvOFP genes ranged from 219 to 1245 bp, encoding the proteins with 72 to 414 amino acid (aa) and 8.3 to 47.3 kDa in molecular weight (MW). Further sequence analysis showed that the isoelectric points (pI) of the FvOFPs ranged from 4.04 to 9.78. The grand average of hydropathicity (GRAVY), ranging from −0.064 to −1.033, showed that all FvOFPs were hydrophilic proteins. The predictive subcellular localizations of the FvOFPs were all in the nucleus. The detailed information of the FvOFP family is listed in Table 1.

3.2. Gene Structure and Encoding Protein Architecture of FvOFPs

FvOFP genes are distributed unevenly on all chromosomes of F. vesca, except for Chromosome 5 (Figure 1A). Based on the deduced protein sequences encoded by the FvOFPs, the 14 genes could be classified into four groups (Figure 1B). The FvOFPs are intron- poor in terms of gene structure, and only FvOFP10 and 12 have one intron. For the protein characters, two conserved motifs were found in all FvOFPs, except for FvOFP2, which did not contain motif 2. The results indicated that motif 1 and motif 2 were conserved domains of the FvOFP family, located in the OVATE domain (Figure 1C). To elucidate the architectures of FvOFPs, the three-dimensional structures of the conserved OVATE domains were, respectively, predicted by the Phyre2 protein modelling server. The results showed that the crystal structure of OVATE domains in FvOFPs was most similar to the programmed cell death 4 middle or C-terminal MA-3 domain with three α-helices (Figure 1D).

3.3. Syntenic and Phylogenetic Analysis of FvOFP Genes

The synteny of the genome between F. vesca and Arabidopsis was assayed by MCScanX. The syntenic relationship between FvOFPs and AtOFPs is displayed in Figure 2A. Ten FvOFPs were found to be orthologous with AtOFPs. Among these, eight FvOFPs had only one ortholog, FvOFP3 and AtOFP7, FvOFP4 and AtOFP11, FvOFP6 and AtOFP5, FvOFP7 and AtOFP10, FvOFP9 and AtOFP11, FvOFP10 and AtOFP1, FvOFP12 and AtOFP6, and FvOFP13 and AtOFP14; FvOFP8 had two orthologs as AtOFP7/AtOFP10; FvOFP11 had three orthologs as AtOFP10/AtOFP15/AtOFP18 (Figure 2A). To study the evolutionary relationships of OFP genes in plants, an NJ phylogenetic tree was constructed using 14 FvOFPs and the OFPs from Arabidopsis (18 AtOFPs), tomato (26 SlOFPs), rice (31 OsOFPs), and pepper (26 CaOFPs). As a result, these OFP genes were clustered into five distinct groups in the phylogenetic tree (Figure 2B). Interestingly, Group 2 had the most FvOFPs (five). Group 1 and 4, respectively, had four and three FvOFPs. Meanwhile, the remaining FvOFPs, 11 and 13, were clustered into Group 5. To some extent, the OFP genes belonging to the same branch are more closely related and have similar biological functions.

3.4. Tissue Expression Pattern and Subcellular Localization of FvOFPs

To validate the tissue expression patterns of the genes, the expression of the FvOFPs in various tissues of the strawberry, including roots, stems, and leaves, was determined through RT-qPCR. The results showed that the FvOFPs exhibited different expression patterns in different tissues (Figure 3A). Some genes are expressed specifically in one or a few tissues. FvOFP1, FvOFP5, FvOFP10, and FvOFP13 had relatively higher expression levels in the roots. FvOFP3, FvOFP4, FvOFP5, FvOFP6, and FvOFP9 specifically express in style. The results indicated their special roles in the development of specific organs. Since fruit (receptacle and achene) is the main harvest of strawberries, it was found that most of the FvOFPs were poorly expressed in fruit, except for FvOFP1, FvOFP11, FvOFP12, and FvOFP14, which were highly expressed highly in achene. To verify the results, the expression patterns of these four FvOFP genes were further assayed in fruit at four different developmental stages (green, white, turning, and red). Similarly, very low expression levels of the four FvOFP genes were detected in all the developmental stages of the receptacle, but high expressions were detected in achene. The expressions of FvOFP1, FvOFP11, and FvOFP14 peaked at the early stage of fruit development (green) and were then downregulated. However, FvOFP12 displayed a sustained upregulation from green to the turning stage, but were extremely downregulated in the red stage (Figure 3B).
The homologous genes of FvOFP1, FvOFP11, FvOFP12, and FvOFP14 were also analyzed in cultivated strawberry (F. × ananassa). Since F. × ananassa is octoploid, three to five highly homologous FaOFP genes could be found for each FvOFP. The expression of these FaOFPs was analyzed using the RNA-Seq data of the receptacle and achene at four developmental stages (green, white, turning, and red; Figure 4A). According to the RNA-Seq data of F. × ananassa, FaOFP1s (FaOFP1-1 to 1-3), FaOFP11s (FaOFP11-1 to 11-5), FaOFP12-1, and FaOFP14-1 displayed a similar expression pattern to their homologues in FvOFPs in fruit. However, the other homologous genes, FaOFP12-2, FaOFP12-3, FaOFP14-2 and FaOFP14-3, were not or were very poorly expressed in both receptacle and achene (Figure 4B). The results suggested that some homologous genes evolved differently in the regulation of gene expression and might have diversified functions.
To further confirm the subcellular localization of these four FvOFPs (FvOFP1, FvOFP11, FvOFP12, and FvOFP14), the coding sequences of the four FvOFP genes were fused in-frame with the GFP gene under the control of the CaMV 35S promoter. The fusion constructs (35S::FvOFPs:GFP) and the control construct (35S::GFP) were transiently transformed into tobacco leaves. As a result, the GFP fluorescence expressed from the control construct was dispersed throughout the whole cell. For the four fusion proteins, distinct GFP fluorescence can be observed in the nucleus (Figure 5).

4. Discussion

OVATE family proteins are important plant-specific TFs which are involved in multiple aspects of growth and development [1], especially in the regulation of fruit development and shape [34,35]. OFPs were present in 13 sequenced plant genomes that represent the major evolutionary lineages of land plants [36]. Here, we showed possible collinearity of OFP genes between F. vesca and Arabidopsis, and constructed an NJ phylogenetic tree. Interestingly, Arabidopsis, tomato, rice, and pepper have 18, 26, 31, and 26 OFP genes, respectively (Figure 2B), while F. vesca has only 14 members. In addition, two FvOFPs (FvOFP8 and FvOFP11) were found in more than one ortholog with Arabidopsis (Figure 2A). The results suggest that the FvOFP gene family contracted during the evolution process.
The OFP family is thought to be a plant growth suppressor and is expressed in the reproductive organs in the early stages of flower and fruit development as determined by RT-PCR analysis [7]. According to the expression pattern assayed by RT-qPCR, three of the four genes (FvOFP1, FvOFP11, and FvOFP14) and their homolog genes in F. × ananassa expressed highly in fruit organs (receptacle and achene) especially at the early stage of fruit development (green; Figure 3B and Figure 4B), which was consistent with previous studies. However, FvOFP12 displayed a sustained upregulation from the green stage to the turning stage, as did its homolog FaOFP12-1 (Figure 3B and Figure 4B), which was similar to that of both the tomato cultivar Heinz1706 and the wild tomato S. pimpinellifolium [8]. It might be interesting to further investigate the role of OFPs at the fruit ripening stage. Additionally, FvOFP14 is phylogenetically close to AtOFP1 and AtOFP5 (Figure 2B), which were proven to be essential to male gamete, pollen function, and proper female gametophyte development, interacting with the TALE transcription factors KNAT3 and BELL and transcription factor BLH1 [16,18]. Moreover, AtOFP1 was also found to interact with BLH3 to regulate the vegetative-to-reproductive phase transition in Arabidopsis [37]. According to the tissue expression pattern, FvOFP14 was expressed specifically highly in achenes (Figure 3A). These results imply that FvOFP14 might share common functions with AtOFP1 and AtOFP5 and play a potential role in regulating the development of reproductive organs.
Previous studies have also shown complex pleiotropic effects of OFPs on plant growth and development. Overexpression of AtOFP6, the ortholog of FvOFP1 (Figure 2A), resulted in flat, thick, and cyan leaves [6], while overexpression of AtOFP7, the homolog of FvOFP1 (Figure 2B), resulted in kidney-shaped cotyledons, as well as round and curled leaves [6]. Overexpression of AtOFP15, one of the orthologs of FvOFP11 (Figure 2A), led to another distinct phenotype including blunt-end siliques [6]. AtOFP1, the homolog of FvOFP14 was reported to inhibit gibberellic acid (GA) biosynthesis by suppressing AtGA20ox1 activity, resulting in dwarf, rosette-like leaves [33]. The expression pattern analysis showed that the four FvOFP genes (FvOFP1, FvOFP11, FvOFP12, and FvOFP14) were also moderately expressed in nutritional organs such as leaves, stems, and roots, suggesting that they might have additional functions in regulating plant morphology and growth. It would be interesting to further explore the specific role of these genes in strawberry using genetic means and methods, such as gene, editing in subsequent studies.
TFs function as key regulators during different stages of fruit development [38,39], and most of them function through synergistic or antagonistic effects of various plant hormones. For instance, the AP2/ERF (APETALA 2/Ethylene response factor) family acts downstream of the ethylene signaling pathway and initiates the expression of many ripening-related genes [40]. The auxin response factor (ARF) family is a key component in auxin signal transduction [41]. The OFP gene family was also reported to be involved in hormone signaling, such as ethylene, IAA, GA, JA, and SA [6,15,22,24]. Moreover, AtOFP1 and AtOFP4 were reported to be involved in forming cell walls, the same as the ripening-related TF, the basic helix–loop–helix (bHLH) in banana [42,43]. These results suggest that OFP might also have a role in hormone signaling and share a similar regulating mechanism with some essential TFs in fruit development and ripening. However, only AtGA20ox1 is a direct target of AtOFP1 so far [16,17], making it clear that the direct targets of OFPs would be valuable for discovering new-development-regulating and hormone-signaling pathways that include OFPs at their core. So far, nearly all OFPs have been proven to be predominantly localized in the nucleus, which was consistent with the subcellular location results for FvOFPs (Table 1; Figure 5). And nearly all OFPs with known functions have been found to regulate plant growth and development via interaction with homeodomain proteins, including the TALE transcription factors KNOX and BELL [5]. However, the possibility cannot be excluded that OFPs may regulate plant growth and development in ways other than directly targeting the expression of particular genes or interaction with other TFs [5]. For instance, it was predicted in rice that some of the OFPs may function as potential nucleocytoplasmic shuttling proteins and can be regulated by various signals such as hormones and light [9]. Further analysis such as in situ localization in different plant cells is needed to understand the role of OFPs and figure out the inner mechanism of OFPs regulating biological processes in fruit development and ripening.

5. Conclusions

In this study, we performed a genome-wide investigation of the OFP gene family in the wild diploid strawberry (F. vesca), and analyzed the physical and chemical properties, gene structure, phylogeny, expression patterns, and subcellular localization of these genes. A total of 14 putative strawberry OFP genes (FvOFPs) were identified. Collinearity analysis showed ten pairs of collinearity between F. vesca and Arabidopsis. Phylogenetic analysis divided FvOFP genes into five different clades. The gene expression patterns of the FvOFP gene family were assayed in different tissues by the RT-qPCR, suggesting that four strawberry OFP genes (FvOFP1, FvOFP11, FvOFP12, and FvOFP14) could play versatile roles in the development of fruit. Subsequently, we focused on these genes and further verified their expression in the fruits at different developmental stages of F. vesca, as well as their homologous genes in the cultivated strawberry (F. × ananassa). Additionally, the subcellular localization of FvOFP1, FvOFP11, FvOFP12, and FvOFP14 were analyzed in the tobacco leaves. The fusion proteins showed distinct GFP fluorescence in the nucleus. The present findings may lay the foundation for further studies to unravel the functions of strawberry OFP genes in fruit ripening, plant growth, and development.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy14030569/s1. Supplementary Data S1—Protein sequences of OFPs from plants. Supplementary Data S2—Sequences of primers used in this study. Supplementary Data S3—Sequence alignment of FvOFPs. Supplementary Data S4—CDS sequences of FvOFP gene family. Supplementary Data S5—Protein sequences of FvOFP gene family.

Author Contributions

Conceptualization, X.C. and B.M.; methodology, X.X., X.W., S.Z. and X.H. writing, X.X. and X.W.; editing and supervision, X.C. and P.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant numbers: 32071987 and 32101697) and the Natural Science Foundation of Zhejiang Province (grant numbers: LQ22C130005, LZ23C130004, and LZ24C130016).

Data Availability Statement

All data related to this study are open-access, and the databases, websites, and software information used have been detailed in the article and are available for interested researchers.

Acknowledgments

The authors thank Zhihong Zhang from Shenyang Agricultural University for kindly providing the seeds of strawberry Rugen (F. vesca).

Conflicts of Interest

The authors declare no conflicts of interest.

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Agronomy 14 00569 g001
Figure 1. Sequence characters of FvOFP genes and their encoding proteins. (A) Chromosomal localization and distribution of OPF genes in F. vesca genome. (B) The Phylogenetic tree, exon–intron structures, and motif compositions of FvOFP proteins or FvOFP genes. (C) Amino acid sequence alignment of the OVATE domains from FvOFPs. The conserved residues are boxed in black or light gray based on the degree of conservation. Motifs corresponding to (B) were underlined in blue and red. (D) Tertiary structure of the OVATE domains predicted by Phyre2.
Figure 1. Sequence characters of FvOFP genes and their encoding proteins. (A) Chromosomal localization and distribution of OPF genes in F. vesca genome. (B) The Phylogenetic tree, exon–intron structures, and motif compositions of FvOFP proteins or FvOFP genes. (C) Amino acid sequence alignment of the OVATE domains from FvOFPs. The conserved residues are boxed in black or light gray based on the degree of conservation. Motifs corresponding to (B) were underlined in blue and red. (D) Tertiary structure of the OVATE domains predicted by Phyre2.
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Figure 2. Syntenic and phylogenetic analysis of FvOFP genes. (A) The syntenic relationship of OFP genes between cultivated F. vesca and A. thaliana. The red lines highlighted the syntenic OFP gene pairs. Orange and green represent F. vesca and Arabidopsis thaliana genomes, respectively. The unit of Mb indicated the relative chromosome size. (B) An NJ phylogenic tree of the OFPs using 14 FvOFPs (F. vesca), 18 AtOFPs (A. thaliana), 26 SlOFPs (Solanum lycopersicum), 31 OsOFPs (Oryza sativa), and 26 CaOFPs (Capsicum annuum). The protein sequences and gene accessions (https://bioinformatics.psb.ugent.be/plaza/, accessed on 1 October 2022) were shown in Supplementary Data S5. Four groups were highlighted in different colors. Numbers in the branches of trees are bootstrap values.
Figure 2. Syntenic and phylogenetic analysis of FvOFP genes. (A) The syntenic relationship of OFP genes between cultivated F. vesca and A. thaliana. The red lines highlighted the syntenic OFP gene pairs. Orange and green represent F. vesca and Arabidopsis thaliana genomes, respectively. The unit of Mb indicated the relative chromosome size. (B) An NJ phylogenic tree of the OFPs using 14 FvOFPs (F. vesca), 18 AtOFPs (A. thaliana), 26 SlOFPs (Solanum lycopersicum), 31 OsOFPs (Oryza sativa), and 26 CaOFPs (Capsicum annuum). The protein sequences and gene accessions (https://bioinformatics.psb.ugent.be/plaza/, accessed on 1 October 2022) were shown in Supplementary Data S5. Four groups were highlighted in different colors. Numbers in the branches of trees are bootstrap values.
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Figure 3. Gene expression analysis of OFP genes in F. vesca. (A) Expression patterns of OFP genes in different tissues including the roots, stems, leaves, petals, anthers, and styles of 70-day-old plants, as well as the receptacles and achenes during the green fruit stage. (B) Expression patterns of FvOFP1, FvOFP11, FvOFP12, and FvOFP14 genes in different stages of receptacle and achene.
Figure 3. Gene expression analysis of OFP genes in F. vesca. (A) Expression patterns of OFP genes in different tissues including the roots, stems, leaves, petals, anthers, and styles of 70-day-old plants, as well as the receptacles and achenes during the green fruit stage. (B) Expression patterns of FvOFP1, FvOFP11, FvOFP12, and FvOFP14 genes in different stages of receptacle and achene.
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Figure 4. Expression patterns of OFP genes in different stages of achene and receptacle in F. × ananassa. (A) The fruit of F. × ananassa in different developmental stages such as green, white, turning, and red. (B) Expressional heatmap of OFP genes based on the RNA-Seq data of different developmental stages of achene and receptacle in F. × ananassa.
Figure 4. Expression patterns of OFP genes in different stages of achene and receptacle in F. × ananassa. (A) The fruit of F. × ananassa in different developmental stages such as green, white, turning, and red. (B) Expressional heatmap of OFP genes based on the RNA-Seq data of different developmental stages of achene and receptacle in F. × ananassa.
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Figure 5. Subcellular localization of the four FvOFPs in tobacco leaves. Green fluorescent protein (GFP) is the control, FvOFPs:GFP are fusion proteins (Bar = 50 μm).
Figure 5. Subcellular localization of the four FvOFPs in tobacco leaves. Green fluorescent protein (GFP) is the control, FvOFPs:GFP are fusion proteins (Bar = 50 μm).
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Table 1. The characteristic parameters of the FvOFP gene family in Fragaria vesca.
Table 1. The characteristic parameters of the FvOFP gene family in Fragaria vesca.
Gene NameGene
Accession
Number 1		CDS (bp) 2Protein (aa) 3Protein Physicochemical
Parameters		Predicted
Subcellular
Localization 6
MW (kDa) 4pIGRAVY 5
FvOFP1FvH4_1g04770.t1101133638.59.78−0.699Nuclear
FvOFP2FvH4_1g09620.t1219728.35.20−0.064Nuclear
FvOFP3FvH4_1g13640.t199933237.89.64−0.729Nuclear
FvOFP4FvH4_2g23310.t168722825.25.30−0.355Nuclear
FvOFP5FvH4_3g01350.t153117620.87.00−0.842Nuclear
FvOFP6FvH4_3g07180.t1117339044.59.33−1.033Nuclear
FvOFP7FvH4_3g25970.t163621123.86.16−0.683Nuclear
FvOFP8FvH4_3g40310.t1115838544.09.67−0.9611Nuclear
FvOFP9FvH4_4g15660.t172324027.05.06−0.652Nuclear
FvOFP10FvH4_6g10140.t1124541447.39.68−0.921Nuclear
FvOFP11FvH4_6g19870.t189429732.44.04−0.579Nuclear
FvOFP12FvH4_6g19871.t180126630.09.44−0.336Nuclear
FvOFP13FvH4_6g31210.t189729834.36.38−0.907Nuclear
FvOFP14FvH4_7g33090.t131810512.05.08−0.269Nuclear
1 Accession of OFP genes in F. vesca genome in GDR database (V4.0.a2, https://www.rosaceae.org/, accessed on 1 October 2022); 2 code sequence (CDS) length of genes; 3 Amino-acid number of proteins; 4 grand average of hydropathicity (GRAVY) scores; 5 molecular weight (MW) of proteins; 6 subcellular localization of proteins predicted by program Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/, accessed on 1 October 2022).
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Xu, X.; Wang, X.; Zhou, S.; Huang, X.; Liu, P.; Ma, B.; Chen, X. Genome-Wide Identification and Characterization of the OFP Gene Family in the Wild Strawberry Fragaria vesca. Agronomy 2024, 14, 569. https://doi.org/10.3390/agronomy14030569

AMA Style

Xu X, Wang X, Zhou S, Huang X, Liu P, Ma B, Chen X. Genome-Wide Identification and Characterization of the OFP Gene Family in the Wild Strawberry Fragaria vesca. Agronomy. 2024; 14(3):569. https://doi.org/10.3390/agronomy14030569

Chicago/Turabian Style

Xu, Xiao, Xinyu Wang, Sirui Zhou, Xumo Huang, Pengcheng Liu, Bojun Ma, and Xifeng Chen. 2024. "Genome-Wide Identification and Characterization of the OFP Gene Family in the Wild Strawberry Fragaria vesca" Agronomy 14, no. 3: 569. https://doi.org/10.3390/agronomy14030569

APA Style

Xu, X., Wang, X., Zhou, S., Huang, X., Liu, P., Ma, B., & Chen, X. (2024). Genome-Wide Identification and Characterization of the OFP Gene Family in the Wild Strawberry Fragaria vesca. Agronomy, 14(3), 569. https://doi.org/10.3390/agronomy14030569

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