The wild diploid strawberry F. vesca accession Heilongjiang-3 was obtained from the strawberry germplasm resource greenhouse of the College of Horticulture, Northwest A&F University, Shaanxi, Yangling, China (34° 20′ N 108° 24′ E). The potted strawberry plants were grown at 22°C with 75% relative humidity and no supplemental light. ‘Heilongjiang-3’ strawberry organs/tissues (roots, stems, runners, leaves, floral buds, flowers, mature flowers with partially withered petals, mature green receptacles, white receptacles with green achenes, half white and half red fruits, and fully ripened fruits) were collected for tissue-specific and different developmental stages of the fruits were collected for stage-specific transcript analysis of the FvTCP genes. The strawberry tissue culture plantlets were transferred to proliferation medium consisting of an MS basal medium supplemented with 30 g L^-1 sucrose, 7 g L^-1 agar, 0.2 mg L^-1 6-benzyladenine (6-BA) and 0.8 mg L^-1 indole- 3-butyric acid (IBA) with monthly subculturing for induction and the following five different subcultural propagation periods (P1: original plantlet; P2: plantlet with 1–2 branch crowns, approximately 2 weeks after subculture; P3: plantlets with 3–4 branch crowns, approximately 3 weeks after subculture; P4: plantlets with 5–7 branch crowns, approximately 4 weeks after subculture; P5: plantlets with over 10 branch crowns, approximately 6 weeks after subculture) were harvested. Arabidopsis thaliana ecotype Col-gl was grown at 22°C with 75% relative humidity under short-day (8 h of light at 125 μmol⋅m^-2⋅s^-1 and 16 h of dark) conditions for 4–5 weeks before transformation.

Six-month-old uniformly developed strawberry plantlets were selected for the stress treatments. Salt stress was simulated by irrigating potted strawberry plants with 300 mM NaCl. Another set of control ‘Heilongjiang-3’ plantlets was similarly treated with distilled water. Cold and heat stress treatments were performed by transferring the plants to a 4°C/42°C chamber for 48 h. Another set of potted ‘Heilongjiang-3’ plantlets was maintained in the control temperature range from 22 to 27°C. Hormone treatments were performed by spraying the strawberry leaves with a solution containing 0.1 mM abscisic acid (ABA), 1 mM salicylic acid (SA), 0.1 mM methyl jasmonate (MeJA), or 0.5 g/L ethephon (Eth), while another set of control ‘Heilongjiang-3’ plantlets were similarly sprayed with distilled water. The leaves of all of the above plants treated with salt, cold, heat, and hormone stresses were then collected at 0, 0.5, 2, 4, 8, 12, 24, and 48 h post-treatment (hpt). Drought stress treatment was performed by withholding water, followed by sampling at 0, 24, 48, 72, 96, 120, and 144 hpt. The plants were watered again after 144 h of drought stress and sampled after another 24 h. Strawberry plantlets grown without drought stress were used as a control. The powdery mildew (Podosphaera aphanis) treatment experiment was conducted by touching the adaxial epidermis with sporulating colonies on the surface of the strawberry leaves. The inoculated leaves were collected at 0, 24, 48, 72, 96, 120, 144, and 168 h post-inoculation (hpi), and uninfected leaves served as a negative control. At each time point of each treatment, six leaves from six separate plants were combined to form one sample, and all of the treatments were evaluated in triplicate. All of the collected plant samples were immediately frozen in liquid nitrogen and stored at –80°C until use.

To identify TCP genes in strawberry, we downloaded the full-length amino acid sequences of hypothetical TCP transcription factor in the diploid woodland strawberry (F. vesca) accession Hawaii-4 from the Plant Transcription Factor Database¹. Next, the full-length amino acid sequences of the hypothetical TCP proteins were employed as query to perform BLAST-P searches in National Center for Biotechnology Information (NCBI) database². An E-value of 1e-10 was used as the threshold to ensure the discovery of all potential TCP DNA binding domain-encoding sequences in the strawberry genome (F. vesca Annotation Release 101). Finally, the putative TCP genes were manually verified using the InterProScan program³ and Pfam database⁴ to validate the presence and completeness of a TCP DNA-binding domain (PF03634). The identified strawberry TCP genes were annotated based on their respective linkage group distribution. Linkage group assignments were retrieved from annotations downloaded from the NCBI Map Viewer⁵. FvTCP genes were mapped to the linkage groups using MapInspect software. The TCP protein sequences from Arabidopsis thaliana, apple, rice, and tomato were retrieved from the Plant Transcription Factor Database (PlantTFDB⁶).

Multiple sequence alignments of TCP proteins and domains were performed separately using ClustalX 2.0.12, and the alignment results were presented and manually modified using GeneDoc. Target prediction for miR319 was performed using the psRNATarget online tool⁷. The full-length amino acid sequences of the putative TCP proteins from Arabidopsis (AtTCP), apple (MdTCP), strawberry (FvTCP), tomato (SlTCP), and rice (OsTCP) were used to generate a phylogenetic tree based on MUSCLE alignment and the unrooted neighbor-joining method with MEGA 5.0. Neighbor-joining analysis with pairwise deletion and bootstrap analysis with 1000 replicates was performed using the p-distance model (Wei et al., 2016).

TCP protein sequences in F. vesca were submitted to the online Multiple Expectation maximization for Motif Elicitation (MEME⁸) program to identify conserved protein motifs. The optimized MEME parameters were as follows: minimum motif width, 6; maximum motif width, 100; and maximum number of motifs, 12. The FvTCP genomic sequences and CDS sequences extracted from NCBI were compared using the online program GSDS 2.0⁹ to infer the exon/intron organization of the FvTCP genes.

FvTCP nucleotide sequences were used to retrieve whole genomic sequences from NCBI using their gene IDs¹⁰. The upstream 1 kb region of the translation start site of the FvTCP genes was used for putative promoter cis-acting element analysis in PlantCARE¹¹. The motifs putatively involved in plant growth and development, phytohormone responses, and abiotic and biotic stress responses are summarized.

To describe the transcript accumulation profiles of TCP genes in F. vesca, total RNA was extracted from tissue samples or treated leaves using an EZNA Plant RNA Kit (R6827-01, Omega Bio-tek, USA), according to the manufacturer’s protocol. Prior to reverse-transcription, RNA was treated with DNase I (RNase free; TaKaRa Biotechnology, Dalian, China) to remove any residual contaminating genomic DNA. Next, 1.5 μg of total RNA isolated from each sample was reverse-transcribed into first-strand cDNA using PrimeScript RTase (TaKaRa Biotechnology, Dalian, China). Gene-specific primers for each FvTCP gene were designed using VECTOR NTI.

Semi-quantitative reverse-transcription (RT) PCR reactions were conducted using the following profile: 95°C for 3 min, 29–33 cycles of 95°C for 30 s, 55°C for 30 s and 72°C for 30 s, and a final step at 72°C for 5 min. The PCR products were separated in a 1.5% (w/v) agarose gel, stained with ethidium bromide, and imaged under UV light for further gene transcript analysis. Each reaction was performed in triplicate, with three independent analyses for each treatment showing the same trends for each gene. The transcript profiles obtained by semi-quantitative RT-PCR were collated and analyzed using GeneSnap software. The heat maps and hierarchical clustering of gene transcript data were visualized in Multi Experiment Viewer (MeV) 4.9 software (Saeed et al., 2006; Guo et al., 2014). Reverse transcription quantitative PCR (RT-qPCR) was performed as described previously with some modifications (Wei et al., 2016). We performed RT-qPCR in a 21-μl reaction volume containing 10.5 μl of SYBR Green Premix Ex Taq II (TaKaRa Biotechnology), 1.0 μl of cDNA, 0.8 μl of 1.0 μM of each primer, and 7.4 μl of sterile distilled H₂O, using an IQ5 real time-PCR machine (Bio-Rad, Hercules, CA, USA). RT-qPCR amplification was performed under the following conditions: 40 cycles at 95°C for 30 s and 58°C for 30 s. After amplification, the samples were maintained at 50°C for 1 min, and the temperature was gradually increased by 0.5°C every 10 s to perform the melting curve analysis. The interspacer 26S-18S strawberry RNA gene (housekeeping gene; Fv18S) was used as an internal control to normalize the expression data (Raab et al., 2006; Hu et al., 2015; Medina-Puche et al., 2015; Paniagua et al., 2016; Wei et al., 2016). Three biological replicates with three technical replicates were assayed for each sample. Reactions for the reference gene were included in each plate. The relative transcript levels of the genes were calculated using the 2^-ΔΔCt method, and the standard deviation was calculated from three biological replicates. The gene-specific primers are listed in Supplemental Table S1.

Based on the public NCBI database (F. vesca Annotation Release 101; Darwish et al., 2015), we designed 10 pairs of gene-specific primers to clone the randomly selected 10 TCP genes in the diploid woodland strawberry F. vesca accession Heilongjiang-3. The predicted coding sequences of the selected FvTCP genes were amplified with high-fidelity HS polymerase (TaKaRa Biotechnology, Dalian, China) using the primers listed in Supplementary Table S1. Finally, six FvTCP genes (FvTCP7, FvTCP8, FvTCP9, FvTCP13, FvTCP15, and FvTCP17) belonging to different types were successfully cloned from the cDNA of the leaves of ‘Heilongjiang-3’.

To construct GFP-tagged FvTCP, the six cloned FvTCP sequence-encoding genes were cloned into the XbaI and KpnI sites in the pBI221 vector. The plasmid DNA of pBI221 containing 35S::FvTCPs-GFP was introduced into Arabidopsis mesophyll protoplasts using the PEG-Ca²⁺-mediated transformation method (Yoo et al., 2007; Wei et al., 2016). The localization of the fusion protein was observed using an Olympus BX-51 inverted fluorescence microscope (Olympus, Japan). GFP fluorescence, the bright field image, and the red autofluorescence of chloroplasts from the protoplast expression assay were imaged simultaneously and merged together. All transient expression assays were repeated at least three times.

To produce a YFP-tagged FvTCP9, the coding sequence of FvTCP9 was cloned in-frame into the BamHI sites of the binary expression vector C15 to generate plasmid 35S::FvTCP9-YFP. The recombinant plasmids 35S::FvTCP9-YFP and 35S::YFP (C15 empty vector) were transfected into Agrobacterium tumefaciens strain GV3101 via electroporation. C15 empty vector and Agrobacterium tumefaciens strain GV3101 served as the control. Agrobacterium-mediated transient assays were performed as described previously with some modifications (Han et al., 2015). The Agrobacterium suspension was evenly injected throughout the entire Fragaria × ananassa Duch. ‘Toyonoka’ strawberry fruit while it was still attached to the plant, at approximately 12 days after anthesis, using a sterile 1-ml hypodermic syringe. The fruits were harvested for Western blot analysis and ripening-related genes transcript analysis at 3 days after infiltration, and the receptacles were frozen in liquid nitrogen and stored at –80°C until use. For each construct, five uniformly sized fruits were used in the infiltration experiment, and the experiment was repeated three times. The primers used for RT-qPCR analysis of ripening-related genes are listed in Supplemental Table S3 (Han et al., 2015).

For Western blot analysis of FvTCP9 protein, total protein was extracted from infiltrated fruits as described previously with some modifications (Wang et al., 2006). The protein was fractionated by 12% SDS-PAGE and blotted onto PVDF membranes (Millipore) using a semi-dry blotting apparatus as described by the manufacturer (Bio-Rad). GFP was detected using a polyclonal mouse anti-GFP antibody (1:5000 dilution; TransGen Biotech, Beijing, China) and a goat anti-mouse IgG secondary antibody (1:10000 dilution; TransGen Biotech, Beijing, China), according to the manufacturer’s instructions. Proteins separated by SDS-PAGE were detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).

Statistical significance was determined using a paired Student’s t-test¹². The mean ± standard deviations from the mean (SD) of at least three replicates are presented, and significant differences relative to controls are indicated at ^∗∗p < 0.05 and ^∗p < 0.01.

A total of 21 strawberry TCP members were originally obtained via a BLAST-P search in NCBI using the 18 strawberry TCP amino acid sequences that were predicted in the sequenced diploid woodland strawberry genome accession Hawaii-4¹³ (Shulaev et al., 2011; Darwish et al., 2015). Subsequently, to verify the reliability of the initial results, a survey was conducted to confirm the presence of the conserved TCP domain using the InterProScan database¹⁴ and the Pfam database¹⁵. The results showed that two putative strawberry TCP genes (XP_011467829.1 and XP_004298742.1) were discarded because they lacked a conserved TCP domain. As a result, 19 non-redundant FvTCP genes were identified in the diploid woodland strawberry and further annotated as FvTCP1 to FvTCP19 according to their locations in strawberry linkage groups 1–7 (Table 1). The 19 FvTCP genes were unevenly distributed in seven linkage groups, with five FvTCP genes located linkage groups 5 and 6, while none of the FvTCP genes mapped to linkage group 1 (Supplementary Figure S1). Detailed characteristics of the TCP transcription factors in F. vesca are provided in Table 1.

Based on the predicted FvTCP coding sequences, we originally cloned the randomly selected 10 homologous TCP genes in the diploid woodland strawberry F. vesca accession Heilongjiang-3. Finally, six FvTCP genes (FvTCP7, FvTCP8, FvTCP9, FvTCP13, FvTCP15, and FvTCP17) belonging to different types were successfully cloned from the cDNA of the leaves of the F. vesca accession Heilongjiang-3 and submitted to NCBI GenBank (GenBank Accession Numbers KX227709, KX227710, KX227711, KX227712, KX227713, and KX227714, respectively). The CDS sequences of the six cloned FvTCP genes from accession Heilongjiang-3 also shared high identities (≥99%, E-value = 0, data not shown) with the corresponding FvTCPs in the strawberry accession Hawaii-4.

To assess the classification of the FvTCP genes and to gain some insight into the potential function of FvTCP proteins from well-studied TCPs in other plant species, a total of 147 TCP genes, comprising 24 genes from Arabidopsis, 52 from apple, 22 from rice, 19 from strawberry and 30 from tomato, were used to construct a phylogenetic tree (Figure 1). Additionally, to gain a better understanding of the classification of FvTCP members, multiple sequence alignment was performed spanning the approximately 60-amino-acids core TCP domain of all FvTCP amino acid sequences. The phylogenetic analysis and the TCP domain alignment showed that strawberry TCP proteins can be divided into two classes: class I (or PCF) and class II (Figure 1A). The TCP proteins of all five plant species were classified into two major classes (class I and class II; Figure 1B). The most striking difference between these two classes was a four-amino-acid deletion in the basic domain of class I relative to class II proteins (Figure 2A). Further analysis showed that the phylogenetic tree also supported the Arabidopsis and rice described previously in the classification of class II proteins in CIN and CYC/TB1 two subclades (Yao et al., 2007). According to this classification, the CYC/TB1 subclade contains three FvTCP genes (FvTCP6, FvTCP9, and FvTCP14), and the CIN subclade contains six FvTCP genes (FvTCP3, FvTCP4, FvTCP5, FvTCP10, FvTCP13, and FvTCP15; Figure 2A). Outside the TCP domain, the R domain, an approximately 18-residues arginine-rich motif, is conserved and only present in a subset of class II proteins. As shown in Figure 2B, four class II genes (FvTCP6, FvTCP9, FvTCP14, and FvTCP3) contain the R domain, but FvTCP3 is less conserved. Additionally, three CIN subclade genes (FvTCP3, FvTCP5, and FvTCP13) contained the putative miR319 target site and shared the highest sequence similarity with the Arabidopsis and tomato miR319-targeted TCP genes (Figures 1A and 2C).

To obtain a better understanding of the diversification and evolutionary relationships of the TCP protein in F. vesca, the conserved motifs and exon/intron organization of FvTCPs were analyzed. A new phylogenetic tree was constructed using the protein sequences of FvTCPs, which divided the FvTCP proteins into three subclades. As shown in Figure 3 and Supplementary Figure S2, we used the online MEME tool to predict the conserved FvTCP protein motifs, identifying 12 conserved motifs. As expected, all 19 of the FvTCPs demonstrated the presence of a highly conserved TCP domain (motif 1). The conserved R domain (motif 4) was found in four class II FvTCPs. All of the class II FvTCPs were characterized by motif 2 in the N-terminal TCP domain. By comparison, the C-terminal TCP domain of motif 3 was detected in a subset of the class I proteins. In addition, some motifs were exclusively present in a subset of a particular subclade, suggesting that these motifs may contribute to the specific function of those genes in the subclade. For example, motifs 6, 8, and 10 in PCF, and motifs 9 and 11 in CIN (Figure 3).

To gain further insight into the evolutionary relationships among FvTCP genes, we investigated the exon/intron organization of individual FvTCP genes by aligning the cDNA sequences and corresponding genomic DNA sequences. Overall, the FvTCP genes exhibited a highly conserved exon-intron organization: 18 of 19 FvTCP genes had no intron, while only FvTCP9 genes possessed one intron. Additionally, most of the FvTCP genes within the nearby paralogous genes demonstrated very similar exon/intron distribution patterns in terms of the exon length and the intron number (Figure 3).

The analysis of cis-regulatory elements in promoter sequences is an important feature for understanding gene function and regulation. To identify the likely cis-acting elements of the FvTCPs, the promoter regions (1 kb of genomic DNA sequence upstream of the translation start site) of the FvTCP genes were used to search the PlantCARE database (Postel et al., 2002). A series of cis-acting elements involved in plant growth and development, phytohormone responses, and abiotic and biotic stress responses were identified (Supplementary Table S2). As shown in Figure 4, the Skn-1_motif and GCN4_motifs, cis-acting regulatory elements involved in endosperm expression (Washida et al., 1999), were found in the promoters of 16 and 9 FvTCP genes, respectively. The circadian control element (circadian; Anderson et al., 1994) was found in 10 FvTCP genes. Notably, two leaf development-related cis-acting elements (HD-Zip1 and HD-Zip2; Sessa et al., 1993) were found in the FvTCP15 promoter. Additionally, the zein metabolism regulation element (O2 site), meristem expression and specific activation element (CAT-box and CCGTCC-box), seed-specific regulation element and shoot-specific expression element (RY element and as-2-box; Bobb et al., 1997) were also identified in the promoters of the FvTCP genes. In hormone-related cis-acting elements, the SA-responsive element (TCA element; Goldsbrough et al., 1993), the MeJA-responsive element (CGTCA motif and TGACG motif; Rouster et al., 1997), and the ABA-responsive element (ABRE; Shen and Ho, 1995) were found in the promoters of 11, 8, and 9 FvTCP genes, respectively. The gibberellin-responsive element (GARE motif and P-box; Kim et al., 1992; Washida et al., 1999) and the auxin-responsive element (TGA element, AuxRR core and TGA box; Ulmasov et al., 1997) were observed in 10 and 4 FvTCP genes. Plenty of hormone-responsive elements were identified in the FvTCP promoter sequences, indicating that phytohormones could play crucial roles in the regulation of plant growth and development (Figure 4B). In addition, some stresses-related (e.g., drought, extreme temperatures, salinity, and disease) cis-acting elements were also found in the putative promoter regions of the FvTCP genes (Figure 4).

To investigate the tissue-specific transcript accumulation profiles of TCP genes in F. vesca, we analyzed the transcripts of FvTCP genes using semi-quantitative RT-PCR and validated the results via RT-qPCR analysis of different tissues, including roots, stems, runners, leaves, flowers, floral buds, and fruits (fully ripened fruits) from the diploid woodland strawberry accession Heilongjiang-3. As indicated in Figure 5, some FvTCP genes exhibited tissue-specific transcript accumulation patterns, while other FvTCP genes showed similar transcript accumulation patterns in different tissues, potentially indicating the functional divergence of FvTCP genes during strawberry growth and development. For example, FvTCP6, FvTCP8, FvTCP9, and FvTCP15 were constitutively expressed in every tissue tested at relatively high transcript levels, whereas FvTCP1, FvTCP2, and FvTCP18 were expressed at very low level in all tested tissues (Figure 5A). In contrast, the transcript accumulation levels of FvTCP3 and FvTCP5 were very high in runners, young leaves, and floral buds and they were relatively low in flowers, indicating that they might play an important role in the development of runners, young leaves, and floral buds. A similar transcript accumulation pattern was observed for FvTCP13 (Figure 5B). In particular, FvTCP19 displayed extremely high relative transcript levels in stems, which suggested that it might play a role in the development of strawberry stems. FvTCP4 and FvTCP14 were preferentially expressed at high levels in runners, young leaves, or floral buds and at almost undetectable levels in other tissues (Figure 5B). Additionally, the transcript accumulation patterns of certain genes varied among the eight tissues. For example, FvTCP12 and 17 exhibited high transcript levels in roots, runners, and fruits, but relatively low transcript levels in young leaves, mature leaves, flowers, and floral buds, and FvTCP7, which was abundantly expressed in roots, runners, and floral buds, displayed lower levels in other tissues (Figure 5B). Remarkably, RT-qPCR analysis showed that some FvTCP genes (FvTCP9, FvTCP10, and FvTCP17) were highly expressed in fruits (fully ripened fruits), which indicated that they might play an important role in fruit ripening (Figure 5B). These results prompted us to investigate the transcript of FvTCP genes during various fruit development and ripening stages.

To investigate fruit development and ripening-related strawberry FvTCP genes, we focused on the transcript accumulation patterns of FvTCP genes in fruits during five different developmental stages (mature flowers with partially withered petals, mature green receptacles, white receptacles with green achenes, half white and half red fruits, and fully ripened fruits). Hierarchical clustering was used to describe the various relative levels of FvTCP gene transcripts, which could be differentiated into two distinct groups. As shown in Figure 6A, 11 FvTCP genes were down-regulated during different fruit developmental stages, while five FvTCP genes exhibited up-regulated transcript accumulation patterns. Additionally, three FvTCP genes showed stable transcript accumulation patterns. Among the 16 down-regulated and up-regulated FvTCP genes, we selected 15 relatively dramatically down-regulated or up-regulated FvTCP genes by RT-qPCR to further test their transcript abundance in the various developmental stages of the fruit (Figure 6B). Among them, five TCP genes (FvTCP5, FvTCP7, FvTCP11, FvTCP12, and FvTCP16) exhibited high transcript accumulation patterns in mature flowers with partially withered petals, and three TCP genes (FvTCP3, FvTCP13, and FvTCP17) showed high accumulation patterns in mature flowers with partially withered petals and mature green receptacles in comparison with the other stages (Figure 6B). These data indicate that the eight FvTCP genes may be involved in strawberry fruit development. The transcripts of four genes (FvTCP6, FvTCP10, FvTCP14, and FvTCP19) were gradually upregulated during the ripening process, which suggested that they might function in strawberry fruit ripening (Figure 6B).

To provide additional information on the growth and developmental functions of TCP genes in strawberry, we investigated their transcript accumulation patterns during five different periods of subcultural propagation in strawberry ‘Heilongjiang-3’ using semi-quantitative RT-PCR (Supplementary Figure S3A). In general, the transcript accumulation patterns obtained for the FvTCP genes could be classified into two types (Supplementary Figure S3B). The majority of the FvTCP genes, specifically FvTCP2, FvTCP4, FvTCP6, FvTCP10, FvTCP11, FvTCP12, and FvTCP14, exhibited downregulation from P1 to P5 stage. The remaining FvTCP genes were only upregulated during specific propagation periods. For example, FvTCP8, FvTCP9, FvTCP13, FvTCP18, and FvTCP19 were upregulated at P2 stage, which indicated that they might promote growth and development during early subcultural propagation. Additionally, the transcript levels of FvTCP1, FvTCP9, and FvTCP18 revealed an upregulation at P5 stage, suggesting that these genes might be involved in rooting and bud germination after subculture. FvTCP17 displayed high transcript levels from P2 to P4 stage, implying that FvTCP17 might play vital biological roles in the subcultural propagation processes of whole strawberry.

To determine the potential roles of the FvTCP genes during plant responses to various environmental stresses, semi-quantitative RT-PCR was performed for the 19 FvTCP genes in the leaves of F. vesca plantlets exposed to cold, heat, drought, salt, and powdery mildew treatments. Overall, the FvTCP genes responded to drought and salt treatment to a greater extent than to temperature and biotic treatment (Figure 7). Among them, 10 FvTCP genes were upregulated in response to drought stress, and 13 FvTCP genes responded to salt treatment. In contrast, a minority of the FvTCP genes were only slightly upregulated in response to temperature and biotic treatment (Figure 7). Notably, FvTCP14 and FvTCP19 showed different degrees of upregulation in response to cold, heat, drought, salt, and powdery mildew treatments. The transcript levels of FvTCP4, 7, and 10 responded to at least three treatments (Figure 7).

Plant hormones such as SA, MeJA, ethylene (Eth) and ABA have well-established roles in plant stress signaling networks and developmental processes (Bari and Jones, 2009). To understand how FvTCP gene transcripts accumulate in response to plant hormone treatment, semi-quantitative RT-PCR and RT-qPCR were used to analyze FvTCP transcripts in response to ABA, ethephon (Eth), MeJA and SA in the leaves. For ABA treatment, the transcript levels of 12 FvTCP genes were prominent and rapidly increased to significantly high levels at 0.5 hpt, which were maintained throughout the entire treatment period (Figure 7). Notably, the highest transcript levels of FvTCP5, 7, 12, and 17 reached extremely high levels compared with the basal transcript levels after ABA treatment (Figure 8). Conversely, almost all FvTCP was downregulated or remained nearly unchanged relative to basal transcript levels in response to Eth treatment (Figure 7). Additionally, seven and four FvTCP genes were upregulated in response to MeJA and SA, respectively, while the other TCP members were downregulated or exhibited no significant change (Figure 7). It is worth noting that FvTCP4, FvTCP6, FvTCP10, FvTCP13, FvTCP16, and FvTCP18 were upregulated in response to at least two of the hormone treatments (Figure 7).

It is known that TFs regulate the transcription of target genes by binding to specific cis-elements in their promoters and that this binding occurs in the nucleus. To assess the subcellular localization of the FvTCP TFs, the full-length open reading frames (ORFs) without the stop codon of six cloned FvTCP genes were cloned into a vector in-frame with green fluorescence protein (GFP) under the control of the CaMV 35S promoter. The resulting constructs and empty (control) vector were transiently expressed in Arabidopsis mesophyll protoplasts. Fluorescence microscopy revealed that six FvTCP fusion proteins were clearly localized in the nucleus (Figure 9). Notably, FvTCP7-GFP (ii) and FvTCP17-GFP (ii) were also localized in the nucleus and cytoplasm (Figure 9). To confirm the nuclear and cytoplasm localization of FvTCP7 (ii) and FvTCP17 (ii), we also examined the subcellular localization of FvHsfC1a, a strawberry heat shock transcription factor that has been reported to localize to both the nucleus and cytoplasm of Arabidopsis mesophyll protoplasts (Hu et al., 2015). FvTCP7 (ii), FvTCP17 (ii), and FvHsfC1a exhibited similar subcellular localizations (Figure 9). Additionally, FvTCP7-GFP (iii) and FvTCP17-GFP (iii) fluorescent signals showed a punctate pattern in the cytoplasm that resembled mitochondria (Figure 9).

Agrobacterium-mediated transient gene expression is a rapid and powerful tool for the analysis of gene function in plants. For a more detailed analysis of the biological roles of the FvTCP genes during strawberry fruit development and ripening, we cloned the FvTCP9 gene into the BamHI sites of the binary expression vector C15 and transiently over-expressed it in Fragaria × ananassa Duch. ‘Toyonoka’ attached fruits (12 days after anthesis) (Figure 10A). The Western blot results revealed single protein bands of the expected size (∼75 kDa for FvTCP9-YFP and ∼25 kDa for YFP) in the agroinfiltrated strawberry fruits, whereas antigen-specific bands were not detected in protein extracts from agroinfiltrated Agrobacterium strain GV3101 control fruits (Figure 10B). These results clearly demonstrated the stable integration and protein expression of FvTCP9 in agroinfiltrated strawberry fruits. Additionally, several strawberry fruit ripening-related genes were analyzed by RT-qPCR in the injected fruits. As shown in Figure 10C, transient over-expression of FvTCP9 markedly up-regulated the expression of a series of genes implicated in fruit color and aroma metabolism, including FaCHS (chalcone synthase), FaF3H (flavanone 3-hydroxylase), FaUFGT (UDP-glycose flavonoid 3-O-glycosyltransferase), and FaQR (quinine oxidoreductase). Simultaneously, transient over-expression FvTCP9 dramatically affected the expression of a series of genes implicated in fruit softening, such as FaPE (pectinesterase), FaPG (polygalacturonase), FaCEL (cellulose), FaGAL1/2 (b-galactosidase1/2), FaXYL1 (b-xylosidase1), and FaEXP1/2/5 (expansin1/2/5).

A total of 19 FvTCPs were identified based on the diploid woodland strawberry genome (accession Hawaii-4; Shulaev et al., 2011; Darwish et al., 2015). This number is highly conserved among Arabidopsis (24 members) and rice (22 members; Figure 1B) (Riechmann et al., 2000; Yao et al., 2007). However, it is significantly lower than that present in apple (52 members; Figure 1B), which is consistent with the genome sizes of apple (∼742.3 Mb in M. domestica; Velasco et al., 2010), indicating that TCP genes in different plants have expanded to differing degrees. Sequence alignment and phylogenetic analysis of FvTCP proteins has resulted in their classification into three major subclasses (I, II, and III), with FvTCP genes distributed across all three subclasses. In addition, each subclass contains TCP genes from Arabidopsis, tomato, apple, and rice. FvTCP genes are more closely related to genes from apple TCP genes, demonstrating that apple and strawberry are Rosaceae and evolved more recently from a common ancestor. These results indicate that although plant TCP genes may be derived from a common ancestor, many have undergone distinct patterns of differentiation with the divergence of different lineages. Moreover, the consistency of the motif compositions of FvTCP proteins as well as the exon/intron structures of most FvTCP genes with phylogenetic subclasses further supported the close evolutionary relationships among FvTCPs as well as the dependability of our phylogenetic analysis, as described previously, in cotton and apple (Ma et al., 2014; Xu et al., 2014).

Accumulating evidence suggests that TCP transcription factors are involved in the regulation of cell growth and proliferation, performing diverse functions in multiple aspects of plant growth and development (Martin-Trillo and Cubas, 2010). The CYC/TB1 clade includes genes that are mainly involved in the development of axillary meristems that give rise to either flowers or lateral shoots. AtTCP1, the closest homolog of CYC in Arabidopsis, is involved in the longitudinal elongation of petioles, rosette leaves, and inflorescent stems. The expression pattern of AtTCP1 is strong in the lower part of the inflorescence stem, the distal region of expanding rosette leaves, and the midrib of the blade and petiole during leaf development (Koyama et al., 2010). Additionally, Arabidopsis gain-of-function tcp1-1D mutant plants show elongated leaves and petioles, whereas TCP1-SRDX plants display rounded and epinastic leaves, shortened petioles, and reduced statures (Guo et al., 2010). FvTCP14, which is closely related to AtTCP1, was transcribed at high levels in runners, floral buds, or young leaves and was almost undetectable in other tissues (Figures 1A and 5B). This result is consistent, in part, with the expression profile of AtTCP1 and implies that the FvTCP14 gene in F. vesca functions in runners and in inflorescence and leaf development. BRANCHED1 (BRC1, AtTCP18) and BRANCHED2 (BRC2, AtTCP12), two homologs of TB1 in Arabidopsis, were transcribed at high levels in tissues that mainly contained axillary buds, such as leaf bases and stem, inflorescences, and siliques (Aguilar-Martinez et al., 2007). AtTCP18 acts downstream of auxin and strigolactone to coordinate axillary bud outgrowth, and mutants with reduced activity of either gene show an increased number of rosette branches. In contrast, the up-regulation of AtTCP18 results in an inhibition of lateral branching. AtTCP12 exhibits a weaker or no mutant phenotype compared with AtTCP18 (Aguilar-Martinez et al., 2007; Finlayson, 2007). The phylogenetically close gene of AtTCP18 in strawberry is FvTCP9 (Figure 1A), which displays higher homology with AtTCP18 by NCBI BLAST-P and a significantly higher relative transcript level in runners and fruits (Figure 5). These transcript similarities suggest that FvTCP9 is likely to perform roles similar to AtTCP18 in axillary bud tissue development in strawberry.

Additionally, CIN-like clade genes could be more ancient than the CYC/TB1 clade TCPs and are important for leaf growth and development. In Arabidopsis, five of the CIN subclade members (AtTCP2, 3, 4, 10, and 24) are post-transcriptionally regulated by miRNA319, and miR319 modulates jasmonate biosynthesis, negative leaf curvature, and crinkly leaves, while it positively regulates leaf senescence and affects petal development (Nath et al., 2003; Palatnik et al., 2007; Schommer et al., 2008; Nag et al., 2009). In the present study, the closest strawberry homologs of these Arabidopsis genes are the four FvTCP genes, FvTCP3, FvTCP4, FvTCP5, and FvTCP13, all of which, excluding FvTCP4, carry a putative binding site for miR319 (Figures 1A and 2C). The transcript accumulation levels of FvTCP3, FvTCP5, and FvTCP13 were very high in runners, young leaves, and floral buds. These results indicated that the regulation of leaf growth and development by miRNA-targeted TCP TFs that are homologous to those in strawberry may be consistent in Arabidopsis.

By contrast, Class I FvTCP genes showed more widespread and less tissue-specific transcript accumulation patterns, such as in roots, runners, floral buds, and fruits (Figure 5). These results implied that Class I FvTCP genes might play diverse regulatory roles at multiple growth and development stages. In Arabidopsis, AtTCP14 regulates embryonic growth potential during seed germination, which is related to ABA and GA responses (Tatematsu et al., 2008). In our study, FvTCP7, 11, and 17 were phylogenetically close to AtTCP14 and had similarly high transcript accumulation levels after ABA treatment (Figures 1A and 8). However, FvTCP7, 11, and 17 were down-regulated during different developmental stages of the fruit (Figure 6), suggesting that they were also down-regulated during embryo and seed development. These findings indicate that FvTCP7, 11, and 17 might have functions that differ from those of ABA-mediated embryo and seed development in response to AtTCP14.

Fruit development and ripening is a complex and highly controlled biological process that is controlled by transcriptional regulatory networks involving many transcription factors, such as MADS-box, NAC, and EIN3/EIL (Seymour et al., 2013). In strawberry, the endosperm and seed coat play a crucial role in the fruit set and early stage fruit development (Kang et al., 2013). Promoter analysis showed that most of the FvTCP genes harbored Skn-1_motif and GCN4_motif cis-regulatory elements involved in endosperm expression in their promoters (Figure 4). These results imply that FvTCP genes are likely to play an important role in strawberry fruit development and ripening. In tomato, several SlTCP genes, such as SlTCP12, SlTCP15, and SlTCP18, are preferentially expressed in tomato fruit. Moreover, these genes are regulated by RIN (RIPENING INHIBITOR), CNR (COLORLESS NON-RIPENING), and SlAP2a (APETALA2a) proteins, which are transcription factors with key roles in ripening, suggesting a role during tomato fruit development or ripening (Parapunova et al., 2014). In strawberry, FvTCP12 and FvTCP17, which are homologs of SlTCP15 and SlTCP18, respectively, also showed high transcript accumulation levels in fruits, indicating that FvTCP12 and 17 might play a role in strawberry fruit development or ripening (Figures 1A and 5). Additionally, ethylene is a key regulator during fleshy fruit ripening (Kumar et al., 2014). The current results demonstrate that the majority of FvTCP genes detected herein were downregulated or nearly unchanged following Eth treatment (Figure 7), suggesting that TCP transcription factors regulate strawberry fruit ripening independently of the Eth pathway.

Agro-infiltration of maturing strawberry fruit has been a useful tool for defining the gene contributions to fruit development and ripening (Hoffmann et al., 2006). However, few TCPs have been well characterized in terms of their potential roles in this process. In this study, agro-infiltrated fruits overexpressing FvTCP9 revealed that the expression of a series of ripening-related genes was distinctly up-regulated (more than 3.0-fold), such as FaQR (quinine oxidoreductase), FaCEL (cellulose), and FaEXP1/2/5 (expansin1/2/5). FaQR, an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), is a key flavor compound in strawberries (Raab et al., 2006). FaCEL is involved in strawberry the fruit softening process via the regulation of cellulose degradation (Woolley et al., 2001). Expansins are proteins that have been demonstrated to induce cell wall extension in vitro, and six FaEXP genes have demonstrated that expansions from ripening strawberry fruit are able to catalyze extension (Harrison et al., 2001). Therefore, we speculate that FvTCP9 might play an important role in strawberry fruit development and ripening. However, we must note that FvTCP9 is the only gene that contains an intron in strawberry (Figure 3), and it was quite highly and consistently expressed during strawberry fruit development (Figure 5). Thus, it would be interesting to investigate whether other FvTCP or FvTCP9 homologs in other plants have similar roles in fruit development and ripening.