Genome-Wide Identification and Expression Analysis of Tubby-like Proteins (TLPs) in Fragaria × ananassa Reveals Their Role in Abiotic Stress Responses

TL;DR

This study identifies and analyzes TLP genes in strawberries, showing their role in helping plants cope with drought and salt stress.

Contribution

The study is the first to investigate the role of TLPs in abiotic stress responses in cultivated strawberries.

Findings

Eight FaTLP genes were identified, most retaining key TUBBY and F-box domains.

FaTLP2 and FaTLP7 were strongly induced under drought and salt stress.

Promoter analysis revealed cis-regulatory elements linked to stress and hormone signaling.

Abstract

Background: Cultivated strawberry (Fragaria × ananassa) is one of the most valuable horticultural crops worldwide. Nevertheless, its productivity is increasingly constrained by high susceptibility to adverse environmental conditions, which are intensified by climate change. Drought represents a major limitation, often accompanied by water deficiency and elevated soil salinity. Plants counteract such abiotic stresses through complex molecular defense mechanisms involving transcription factors that regulate stress-responsive gene expression. Methods: In this study, we conducted a systematic bioinformatic analysis of the Tubby-like protein (TLP) transcription factor family in Fragaria × ananassa. RT-qPCR was used to analyze the expression patterns of FaTLP genes under different conditions to elucidate their potential roles in stress adaptation. Results: Eight FaTLP genes were identified in each of the four subgenomes, most of which retained the characteristic TUBBY and F-box domains. Gene expression profiling revealed that several FaTLP genes were differentially expressed in leaves under drought and salt stress, with FaTLP2 and FaTLP7 exhibiting strong induction. In addition, the expression of FaTLP2 and FaTLP7 under various oxidative and signaling-related treatments, as well as in different tissues of strawberry plants were analyzed. Promoter analysis identified multiple cis-regulatory elements associated with phytohormone signaling and abiotic stress responses, such as ABRE, MYB, and MYC motifs. Phylogenetic analysis showed that FaTLP2 and FaTLP7 share high sequence similarity with orthologous TLPs from other plant species known for enhanced stress tolerance, suggesting that these proteins may play conserved roles in the molecular mechanisms underlying drought and salinity resilience. Conclusions: This study provides valuable insights into the potential roles of FaTLPs in regulating environmental signal transduction and transcriptional control, contributing to abiotic stress tolerance in Fragaria × ananassa.

Figures

Gene expression analysis of FaTLP2 (A) and FaTLP7 (B) in different strawberry tissues by RT-qPCR. Relative expression values were calculated in relation to the green fruit receptacle, which was assigned an arbitrary value equal to unity. Values are means ± SEM from 3 biological replicates. Asterisks indicate significant differences (* p < 0.001 and p < 0.01 Student’s t-test).

Full text

1. Introduction

The cultivated strawberry (Fragaria × ananassa), an allo-octoploid species (2n = 8x = 56), originated approximately 300 years ago from the interspecific hybridization between Fragaria chiloensis and Fragaria virginiana. The genomes of these two octoploid progenitors were formed through the fusion and interaction of four diploid genomes over millions of years. The origin of the four subgenomes (A, B, C, and D) of Fragaria × ananassa has therefore been a subject of debate, with several hypotheses proposed. However, current evidence indicates that the diploid species F. vesca and F. iinumae are well-established progenitors of the A and B subgenomes, whereas the origins of the C and D subgenomes remain unresolved. Nevertheless, recent results support earlier reports that F. viridis and F. nipponica are donors of the C and D subgenome [1,2,3,4,5,6]. Since its domestication, the cultivated strawberry has become one of the most important fruit crops worldwide, both economically and nutritionally [2,7]. Its high content of vitamin C, phenolic compounds, and other bioactive metabolites confer strong antioxidant, anti-inflammatory, and cardioprotective properties, enhancing its relevance in the food and nutraceutical industries [8]. Despite its agronomic and nutritional significance, the complex allo-octoploid nature of the F. × ananassa genome continues to pose challenges for genetic and functional studies.

Strawberry plants (F. × ananassa) exhibits high sensitivity to environmental stresses, particularly drought. Drought, characterized by an imbalance between water loss and uptake, impairs key physiological processes such as photosynthesis, cell expansion, and biomass accumulation, ultimately reducing yield and fruit quality [9,10]. To counteract these effects, plants activate complex physiological and molecular defense responses orchestrated by phytohormones and transcription factors (TFs), including members of the NAC, WRKY, MYB, bZIP, and AP2/EREBP families. These factors regulate genes associated with osmotic adjustment, membrane stabilization, and reactive oxygen species (ROS) detoxification [11,12,13].

Within this regulatory network, Tubby-like proteins (TLPs) have emerged as a multifunctional family of transcription factors involved in plant development and stress responses. Initially described in mammals for their role in obesity and neurodegeneration, TLPs were later identified in plants, where they diversified to acquire distinct physiological functions [14,15]. These proteins are characterized by a highly conserved C-terminal TUBBY domain responsible for DNA binding and phosphatidylinositol 4,5-bisphosphate (PIP_2_) interaction and, in most plant species, an N-terminal F-box domain involved in SCF ubiquitin ligase complex formation and ubiquitin-dependent protein degradation [16]. The presence of both domains suggests a functional co-evolution linking transcriptional regulation with post-translational modification.

In model plants, such as Arabidopsis thaliana, Oryza sativa, and Zea mays, TLPs have been implicated in diverse biological processes, including seed germination, organ development, leaf senescence, and, notably, abiotic stress responses [14,15]. In Arabidopsis, several AtTLP members positively regulate drought tolerance [15,17]. Similarly, in fruit crops such as Malus domestica, Solanum lycopersicum and Fragaria vesca, specific TLP genes exhibit differential expression under drought and salinity stress, suggesting roles in osmotic regulation and cellular protection [16,18,19]. Tissue-specific expression patterns further indicate functional diversification among TLP family members.

In cultivated strawberry, genomic studies have enabled the identification of multiple gene families associated with stress tolerance [20,21,22]; however, knowledge regarding TLP genes remains limited. Given the allo-octoploid nature of the F. × ananassa genome, gene redundancy and subfunctionalization may have generated variants with distinct roles in environmental stress response. Therefore, comprehensive analysis of the structure, evolution, and expression of TLP genes is essential for elucidating the molecular mechanisms underlying adaptation to adverse conditions, particularly drought and salinity in strawberry. Such insights may provide a foundation for the development of molecular strategies aimed at enhancing abiotic stress resilience in this economically important crop.

2. Materials and Methods

2.1. Identification and Comprehensive Analysis of the TLP Family in Fragaria × ananassa

Members of the TLP gene family in Fragaria × ananassa were identified using BLASTn searches with coding sequences from Fragaria vesca as queries against the Phytozome database (https://phytozome-next.jgi.doe.gov/blast-search, accessed on 1 November 2024). An E-value cutoff of ≤1 × 10^−5^ was applied to identify putative TLP homologs. Given the allo-octoploid nature of the genome, only orthologs belonging to the A subgenome, which is considered the dominant subgenome compared with the other three subgenomes [12], were retained for further analysis. Molecular weight, theoretical isoelectric point, and coding DNA sequence length, were obtained using the ExPASy server (https://www.expasy.org/, accessed on 5 November 2024). Conserved motifs were identified using MEME (https://meme-suite.org/meme/index.html, accessed on 10 November 2024), and functional domains were verified using the NCBI and Pfam databases (https://www.ebi.ac.uk/interpro/entry/pfam, accessed on 16 November 2024).

Promoter regions (2000 bp upstream of the transcription start site) were extracted from the Phytozome database and analyzed using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 23 November 2024) to identify cis-regulatory elements. Chromosomal localization was determined based on the F. × ananassa ‘Royal Royce’ v1.0 reference genome available in the Genome Database for Rosaceae (GDR; https://www.rosaceae.org/, accessed on 11 January 2025). Both chromosomal localization and the cis-regulatory elements were visualized using TBTools-II v2.154 [23].

Subcellular localization of FaTLP proteins was predicted using Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant/, accessed on 20 January 2025), and cross-validated with CELLO v2.5 (http://cello.life.nctu.edu.tw/, accessed on 20 January 2025). Nuclear localization signals were further analyzed using NLStradamus (http://www.moseslab.csb.utoronto.ca/NLStradamus/, accessed on 21 January 2025). Three-dimensional structures of FaTLP proteins were predicted using the AlphaFold server (https://alphafoldserver.com/, accessed on 15 February 2025) based on the AlphaFold3 server [24].

2.2. Phylogenetic, Collinearity, and Synteny Analysis

Amino acid sequences of TLPs from Fragaria × ananassa and other plant species were retrieved from the Phytozome database using BioMart (https://phytozome-next.jgi.doe.gov/biomart/, accessed on 20 February 2025). Multiple sequence alignments were performed using Clustal Omega (EMBL-EBI; https://www.ebi.ac.uk/jdispatcher/msa/clustalo?outfmt=msf, accessed on 3 March 2025). Phylogenetic trees were constructed in MEGA v11.0 using the maximum likelihood method with 1000 bootstrap replicates. and subsequently edited and visualized using the iTOL platform (https://itol.embl.de/upload.cgi, accessed on 12 March 2025).

Collinearity and synteny analyses were performed using the F. × ananassa ‘Royal Royce’ genome v1.0 and F. vesca genome v4.0.a1, both obtained from the Genome Database for Rosaceae (GDR). The results were visualized using TBTools-II v2.154.

2.3. Plant Material

Strawberry (Fragaria × ananassa cv. ‘Chandler’) plants were grown under controlled conditions at 24–25 °C with a 16 h light/8 h dark photoperiod, using Osram Fuora lamps (Munich, Germany; 120 μmol m^−2^ s^−1^). Prior to the different treatments, plants were transferred to geotextile pots containing half-strength Murashige and Skoog (MS) medium (Duchefa Biochemie B.V., Haarlem, The Netherlands) and acclimated for 24 h.

Drought stress was induced by supplementing half-strength MS medium with 20% polyethylene glycol (PEG) for 5 h under 30% relative humidity, while salt stress was applied using half-strength MS medium containing 200 mM NaCl for 3 days under 70% relative humidity [25]. For ALA (5-Aminolevulinic acid, a regulator of stress tolerance, and SNP (sodium nitroprusside, a nitric oxide donor) treatments, plants were transferred to half-strength MS containing 10 mg L^−1^ ALA and 10 μM NO, respectively [25,26]. Control plants remained in half-strength MS medium. Leaf samples were collected 3 days after treatment.

Hormonal treatments were performed as previously described [27,28]. Briefly, leaves were sprayed to run-off with 100 μM abscisic acid (ABA) or 100 μM methyl jasmonate (MeJA), both dissolved in 5% ethanol containing 0.01% Tween-20. Control plants were sprayed with 5% ethanol containing 0.01% Tween-20. Leaf samples were collected at 6 and 24 h after treatment. Oxidative stress experiments were conducted similarly by foliar spraying with 100 mM H_2_O_2_ or 1 mM paraquat (PQ) dissolved in sterile water containing 0.01% Tween-20 [26]. Leaf samples were collected 5 h after H_2_O_2_ application and at 6 h and 24 h after PQ treatment.

All experiments were performed using three independent biological replicates. Plant material used for the determination of expression profiles in different tissues was obtained from strawberry plants (Fragaria × ananassa cv. ‘Chandler’) grown under greenhouse conditions. Leaves, roots, crowns, stolons, green and red fruits (receptacle), and green and red achenes were collected separately. Following the treatments, leaf samples were harvested immediately. All tissues were flash-frozen in liquid nitrogen and stored at −80 °C until further analysis.

2.4. RNA Isolation and RT-qPCR Analysis

Total RNA was extracted from frozen leaf tissue using the Maxwell^®^ RSC Plant RNA Kit (Promega, Madison, WI, USA), quantified with the QuantiFluor™ RNA System (Promega), and used for first-strand cDNA synthesis with the Reliance Select cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Quantitative real-time PCR (RT-qPCR) was performed on an iCycler iQ Real-Time PCR System (Bio-Rad) using SsoAdvanced™ SYBR^®^ Green SuperMix in 10 μL reactions run in triplicate. Melting-curve analysis (60–95 °C, 0.5 °C increments for 10 s) was included to confirm primer specificity. All RT-qPCR reactions were carried out with three independent biological replicates. Relative expression levels were calculated using the 2^–Δ(ΔCt)^ method [29], with NDR1 and EF1α used as the reference genes [30]. Specific primers for FaTLP genes were designed using NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (accessed 20 March 2025). The primer sequences are listed in Supplementary Table S1.

3. Results and Discussion

3.1. Identification of the TLP Gene Family in Fragaria × ananassa

A comprehensive in silico analysis of the Fragaria × ananassa genome was performed to identify and characterize members of the Tubby-like protein (TLP) family. Coding sequences of TLP genes previously described in Fragaria vesca were used as queries for BLASTn similarity searches, leading to the identification of 32 FaTLP genes. These genes were evenly distributed across the four subgenomes (A, B, C, and D), with eight members in each. The FaTLP genes were located on chromosomes 1 through 6, with the highest gene density on chromosome 2, which contained 3 genes, whereas the remaining chromosomes contained only one gene each. Notably, no FaTLP genes were identified on chromosome 7 (Figure 1). Based on chromosomal location and subgenome assignment, the genes were designated FaTLP1A, B, C, D to FaTLP8A, B, C and D.

Previous studies have reported that the A subgenome is the dominant subgenome in Fragaria × ananassa relative to the other three subgenomes [1]. Given the allo-octoploid nature of the genome, we therefore retained only orthologs belonging to the A subgenome for downstream analyses. Two transcript variants of FaTLP7 (named FaTLP7a and FaTLP7b) were identified, differing by the presence of a 30 bp exon within the coding region, suggesting alternative splicing events and a potential role in post-transcriptional regulation. The coding sequences ranged between 1144 and 1315 bp, encoding proteins of 381–438 amino acids, with predicted isoelectric points between 9.18 and 10.09 and molecular weights of 42.6–49.1 kDa (Table 1). These physicochemical properties are consistent with those reported for TLPs in other plant species, indicating a high degree of structural conservation within the TLP family [14].

Subcellular localization predictions indicated that most FaTLP proteins exhibit dual localization in the nucleus and cytoplasm, consistent with their proposed roles in signal transduction and transcriptional regulation [14]. A subset of FaTLPs was predicted to localize in mitochondria or chloroplasts (Table 1). This distribution suggests that FaTLP1, FaTLP2, FaTLP4, FaTLP6, and FaTLP8 may share functional similarities with Arabidopsis thaliana TLPs, which are known to associate with the plasma membrane via the TUBBY domain and translocate to the nucleus in response to stress stimuli [14,15]. In contrast, the predicted mitochondrial or chloroplastic localization of FaTLP3, FaTLP5, and FaTLP7 may indicate roles in additional biological processes, such as developmental or fruit ripening [31,32] and seed formation [17]. These functions may involve hormone-related signaling pathways, such as jasmonate-associated responses [33], potentially mediated by F-box domain interactions [34]. However, further experimental validation is necessary to elucidate the biological significance of these localization patterns.

The number of TLP genes identified in F. × ananassa (32) is higher than that reported in Arabidopsis thaliana (11), Oryza sativa (14), Malus domestica (9), and Glycine max (22) [14]. This expansion is likely associated with the allopolyploid nature and complex evolutionary history of the cultivated strawberry genome. Collectively, these findings suggest that the expansion of the FaTLP gene family may have provided a basis for functional diversification, potentially contributing to environmental adaptation.

3.2. Phylogenetic, Structural, and Motif Analyses of FaTLPs

Phylogenetic analysis of FaTLP amino acid sequences alongside TLPs from other plant species available in Phytozome database revealed five main clades (Figure 2). FaTLP8 diverged and formed an independent cluster with orthologs from other species. This pattern suggests potential functional specialization or structural divergences. The remaining FaTLPs members clustered into four distinct clades that showed close relationships with TLPs from Malus domestica, Glycine max, Solanum lycopersicum, and Arabidopsis thaliana, indicating a high degree of evolutionary conservation across diverse plant families.

Homology analysis revealed high sequence similarity among FaTLPs, with up to 80% identity between family members and ≥75% identity with orthologs from other species. The highest similarities were observed between FaTLP7 and SlTLP8 (85%) and between FaTLP2 and GmTLP8 (86%), both of which have been previously associated with drought and salinity responses [17,35]. These results support a conserved evolutionary framework for TLP proteins across plant species [14,34]. Domain analysis confirmed the presence of both the conserved TUBBY domain and the N-terminal F-box domain in all FaTLPs, except FaTLP8, which lacks the F-box region (Figure 3A). This structural difference suggests potential functional divergence of FaTLP8, possibly involving pathways independent of ubiquitin-mediated regulation [36].

Motif analysis revealed conserved motif patterns that were largely consistent with the phylogenetic groupings (Figure 3B), indicating that motif diversification accompanied the evolutionary divergence of FaTLP subgroups. Gene structure analysis showed that FaTLP genes contain between 4 and 9 exons (Figure 3C), with FaTLP8 displaying the most complex exon–intron organization.

Predicted 3D structures of FaTLP proteins displayed the canonical β-barrel architecture characteristic of TUBBY domains, with a central α-helix (Figure 4). Notably, FaTLP6 lacked this α-helix, which may affect protein conformation and interactions involved in membrane association or signal perception. Similarly, the absence of the F-box domain in FaTLP8 further supports its structural divergence and suggests potential functional differentiation within the family [37].

These findings indicate that FaTLPs constitute a structurally conserved yet diversified protein family in strawberry, in which variations in gene structure and protein domains may contribute to functional specialization.

3.3. Collinearity and Synteny Between Fragaria vesca and Fragaria × ananassa

Comparative collinearity analysis between F. vesca and F. × ananassa revealed a high degree of syntenic conservation, consistent with their shared evolutionary history, although localized rearrangements were observed (Figure 5). Most FaTLP genes were located within conserved genomic regions. An exception was FaTLP1 from subgenome D, which appears to have originated from a duplication event involving TLP1 and TLP4 in F. vesca.

Gene order was largely conserved, except on chromosome 2C, where the rearrangement (FaTLP4–FaTLP2–FaTLP3) suggests possible inversion or transposition events. In addition, FaTLP7 in subgenome C showed no clear syntenic origin in F. vesca, indicating a potential contribution from other ancestral genomes. These patterns are consistent with the allopolyploid nature of the F. × ananassa genome [4,5]. Overall, the observed synteny suggests that the expansion of the TLP gene family primarily resulted from ancestral duplication events followed by subsequent diversification.

3.4. Promoter Region and Cis-Regulatory Element Analysis

Promoter analysis revealed between 24 and 32 cis-regulatory elements per gene, with FaTLP1 containing the most and FaTLP6 the fewest. Most elements were associated with phytohormone responses, development, and environmental stress. Phytohormone-related motifs included ABA-responsive (ABRE), MeJA-responsive (CGTCA-motif), gibberellin-related (TATC-box, P-box, GARE-motif), salicylic acid-responsive (TCA-element), and auxin-responsive (AuxRR-core, TGA-element) sequences. Light-responsive motifs (G-box, Box-4, GT1, GATA, etc.) were abundant, along with elements linked to seed, meristem, and endosperm development. Stress-related motifs included STRE, TC-rich repeats, MBS, MYC, LTR, and ARE elements (Figure 6).

Heatmap visualization showed that ABRE, MYB, MYC, and ARE motifs were the most abundant across FaTLP promoters (Figure 7). FaTLP1, FaTLP2, and FaTLP4 contained the highest numbers of ABRE elements, suggesting ABA-mediated regulation under drought and salinity stress [38,39,40]. FaTLP8 was enriched in MYB motifs, while FaTLP5 showed a high number of MYC motifs, both of which are associated with drought response and secondary metabolism. FaTLP6 and FaTLP7 lacked ABRE elements but contained MYB/MYC motifs, indicating potential involvement in ABA-independent regulatory pathways [41,42]. In contrast, FaTLP3 was enriched in ARE motifs, suggesting a role in hypoxia-related responses [43]. Overall, these findings indicate that FaTLPs may act as multifunctional regulatory hubs integrating hormonal and environmental signals, consistent with transcription factor-mediated adaptive gene regulation [44].

3.5. Expression Profiling of FaTLPs Under Drought and Salt Stress

To assess the involvement of FaTLPs in responses to water deficit and salt stress, strawberry plants were treated with 20% PEG and 200 mM NaCl. Under both stress conditions, plants exhibited wilting and leaf drying symptoms (Figure 8 and Figure 9). RT-qPCR analysis of leaves from plants exposed to 20% PEG showed significant induction of FaTLP1, FaTLP2, FaTLP3, FaTLP5, and FaTLP7. FaTLP1, FaTLP2, FaTLP5, and FaTLP7 exhibited strong transcriptional induction, with expression levels exceeding 4-fold compared to the control (fold change (FC) values of 5.4, 6.3, 4.4 and 8.5, respectively). In contrast, FaTLP3 displayed a moderate increase in expression, with an FC of 1.8 (Figure 8C).

Under salt stress, FaTLP2, FaTLP5, and FaTLP7 were significantly upregulated. Consistent with their response to drought stress, FaTLP2 and FaTLP7 showed the highest induction levels, with FC values of 3.3 and 6.7, respectively. In contrast to their response under 20% PEG conditions, FaTLP3, FaTLP4, and FaTLP8 were significantly downregulated under high salinity. Moderate repression was observed for FaTLP3 and FaTLP4, with expression levels approximately 2-fold lower than the control (FC = 0.39 and FC = 0.38, respectively), whereas FaTLP8 exhibited much stronger repression, with expression nearly ten-fold lower (FC = 0.01) (Figure 9C). FaTLP6 showed no detectable expression under drought stress, salt stress or control conditions (Figure 8C and Figure 9C). This absence of detectable FaTLP6 expression may indicate very low transcript abundance or tissue-specific regulation. In addition, the lack of stress-related promoter motifs in FaTLP6 suggests the absence of stress-dependent transcriptional regulation under these treatments.

Comparison of expression patterns with promoter analysis revealed a clear correlation between predominant cis-regulatory elements and induction/repression under drought and salinity stress. The induction of FaTLP1, FaTLP2, FaTLP5, and FaTLP7 under drought may be associated with abundant ABRE motifs in FaTLP1 and FaTLP2, and MYC motifs in FaTLP5 and FaTLP7. In contrast, repression of FaTLP4 and FaTLP8 under salt stress appears to be linked to predominant MYB motifs. MYB transcription factors regulate ion transporters such as Na^+^/H^+^ exchangers, SOS1, and high-affinity K^+^ transporters, which maintain ionic homeostasis and mitigate Na^+^ toxicity [45]. The observed repression of these genes in leaves may reflect tissue-specific expression, as many of these transporters function primarily in roots.

MYC transcription factors contribute to drought tolerance in various tissues, including leaves and roots [41], consistent with the induction of FaTLP1, FaTLP2, FaTLP5, and FaTLP7, all containing MYC motifs. This highlights the functional diversity of FaTLP genes and their distinct molecular roles in plant adaptation to different abiotic stresses.

3.6. Expression Analysis of FaTLP2 and FaTLP7

Among the FaTLPs, FaTLP2 and FaTLP7 exhibited the most pronounced differential expression under both drought and salt stress, suggesting that they may play important roles in stress adaptation. Accordingly, we further examined their expression patterns under various stress-related and hormone treatments, as well as in different strawberry tissues.

The transcriptional responses of FaTLP2 and FaTLP7 to ABA and MeJA were evaluated in Fragaria × ananassa leaves using RT-qPCR (Figure 10). Both genes were significantly induced by ABA treatment, with FaTLP7 showing a stronger upregulation than FaTLP2 (FC = 18.1 and FC = 3.2, respectively). MeJA also stimulated the expression of both genes; however, the magnitude of induction was lower than that observed under ABA treatment. Consistently, FaTLP7 exhibited greater induction than FaTLP2 in response to MeJA, with FC values of 6.7 and 2.2, respectively. These results indicate that FaTLP2 and FaTLP7 are responsive to hormone signaling pathways associated with abiotic stress and defense responses in plants.

The transcriptional responses of FaTLP2 and FaTLP7 to ALA, SNP, H_2_O_2_, and PQ were evaluated in Fragaria × ananassa leaves using RT-qPCR (Figure 11). Both genes were significantly induced by oxidative stress treatments, with FaTLP7 showing a stronger upregulation than FaTLP2 under both H_2_O_2_ and PQ treatments, particularly after 24 h of PQ exposure. FaTLP7 expression increased up to 2.4-fold in response to H_2_O_2_ and showed a 4.6-fold induction shortly after PQ treatment, rising to >11-fold after 24 h. In contrast, FaTLP2 exhibited only minor induction at 6 h under H_2_O_2_ and PQ treatments (FC = 1.2 and FC = 1.4, respectively), whereas a marked induction of approximately 4-fold was observed 24 h after PQ exposure. These results suggest that FaTLP7 may play an important protective role under severe oxidative stress conditions. Both genes also displayed similar expression patterns in response to ALA (FC = 1.4 for FaTLP2 and 1.6 for FaTLP7), a regulator of stress tolerance [46], and SNP, a nitric oxide donor [26], which significantly induced transcript accumulation. Notably, SNP treatment resulted in a 2–3-fold increase in expression of both genes, suggesting that FaTLP2 and FaTLP7 may be involved in nitric oxide–dependent signaling pathways.

Tissue-specific expression of FaTLP2 and FaTLP7 was analyzed by RT-qPCR in various strawberry tissues (Figure 12). Both genes were expressed in leaves, roots, crowns, stolons, and fruits, including green and red receptacles and detached achenes (true fruits). FaTLP2 showed the highest transcript levels in achenes at the green fruit stage, showing a 6.3-fold increase relative to green fruit tissue, followed by a decrease at the red stage (FC = 2.8). In contrast, FaTLP7 also exhibited maximal expression in achenes but followed a distinct developmental pattern, with expression increasing slightly from the green (FC= 3.65) to the red stage (FC = 4.1). In addition, both genes were significantly upregulated in leaves and roots, with expression levels exceeding 2-fold relative to the reference tissue (green fruit). Collectively, these results indicate that FaTLP2 and FaTLP7 exhibit tissue-specific and developmentally regulated expression, particularly during achene development.

Gene expression analysis of FaTLP2 (A) and FaTLP7 (B) in different strawberry tissues by RT-qPCR. Relative expression values were calculated in relation to the green fruit receptacle, which was assigned an arbitrary value equal to unity. Values are means ± SEM from 3 biological replicates. Asterisks indicate significant differences (** p < 0.001 and ** p < 0.01 Student’s t-test).*

3.7. Functional Insights into FaTLP2 and FaTLP7

Among the FaTLPs, FaTLP2 and FaTLP7 exhibited the most significant differential expression under both drought and salt stress, identifying them as strong candidates for key roles in stress adaptation. Their promoter regions are enriched in ABRE, MYB, and MYC motifs, consistent with stress-responsive transcriptional regulation. Phylogenetic analysis further supports their functional relevance, revealing close homology between FaTLP2 and GmTLP8 from soybean and between FaTLP7 and SlTLP8 from tomato, species with well-characterized stress-response networks (Figure 13).

Previous studies have demonstrated that overexpression of GmTLP8 [35] and SlTLP8 [16] enhances drought and salt tolerance through multiple mechanisms. These include foliar endoreduplication leading to enlarged pavement cells and reduced stomatal density [47,48], as well as altered root architecture characterized by longer primary roots and increased root hair formation [17,36]. By functional orthology, FaTLP2 and FaTLP7 may perform analogous roles in F. × ananassa, contributing to stress tolerance through coordinated cellular and developmental adaptations.

4. Conclusions

In Fragaria × ananassa, 32 Tubby-like genes (FaTLPs) were identified and found to be evenly distributed across its four subgenomes. Except for FaTLP8, which lacks the F-Box domain, and FaTLP6, which shows variation within the TUBBY domain, all FaTLP proteins possess the characteristic TUBBY and F-Box domains. The proteins share up to 80% identity among themselves and up to 86% identity with orthologs from other species. Most FaTLPs display dual cytoplasmic–nuclear localization, suggesting roles in both environmental signal perception and transcriptional regulation. Synteny analyses indicate strong evolutionary conservation consistent with the allopolyploid origin of cultivated strawberry. Promoter analyses revealed numerous cis-regulatory elements associated with hormonal and abiotic stress responses, in agreement with RT-qPCR profiles showing the induction of FaTLP1, FaTLP2, FaTLP5, and FaTLP7 under drought stress, and of FaTLP2, FaTLP5, and FaTLP7 under salinity, whereas FaTLP4 and FaTLP8 were repressed. Collectively, these findings suggest that FaTLPs act as key regulators of environmental signal transduction and transcriptional control, contributing to abiotic stress tolerance in F. × ananassa.