The strawberry genome and proteome were downloaded from Phytozome V12 (Goodstein et al., 2012) to perform exhaustive data mining of the FveIAA family. The IAA protein sets from A. thaliana were obtained from The Arabidopsis Information Resource (TAIR) (Swarbreck et al., 2008). Default parameters and cutoff value 0.01 was used for the hidden Markov Model (HMM) profiles to identify the Aux/IAA genes from the F. vesca genome (Eddy, 1998).The presence of conserved domains in the candidate Aux/IAA genes was evaluated using PFAM V32 and SMART tools (Finn et al., 2016; Schultz et al., 1998). NCBI CDD was used to examine the uniqueness of the obtained sequences for the Aux/IAA domains. ProtParam software was used to determine the molecular weight (MW) and the isoelectric point (pI) and of the FveIAA proteins. The annotated Aux/IAAs of strawberry were labeled as ‘FveIAA’ followed by a number representing their chromosomal orders.

Based on the strawberry genome database, we mapped all FveIAA genes to strawberry chromosomes using Circos (An et al., 2015). The chromosomal location of the FveIAA family was visualized using MapChart V2.1 (Voorrips, 2002). The gene structure of FveIAA genes was extracted from Phytozome and visualized using GSDS V2.0 (Hu et al., 2015a). MEME V5.1 and PFAM were used to detect the conserved motifs and functional domains, respectively (Bailey et al., 2009; Finn et al., 2016). A Multiple Collinearity Scan toolkit (MCScanX) with default parameters was used to study the gene duplication events (Wang et al., 2013). A syntenic analysis map was built using Dual System Plotter to study the relationship between the orthologous Aux/IAA genes obtained from strawberry and other organisms. STRING V11.0 with a threshold of 0.7 was used to construct the PPI network (Szklarczyk et al., 2017). The promotors (1500 to 1 bp before ATG) of the FveIAA genes were obtained from Phytozome, and the cis-elements were predicted by PlantCARE (Lescot et al., 2002), and subcellular localization of FveIAAs was predicted by WoLF PSORT (Horton et al., 2007).

ClustalW and DNAMAN were used for multiple sequence alignment of the complete FveIAA and AtIAA protein sequences. The neighbor-joining (NJ) phylogenetic tree was built using MEGA V7.0 (Kumar et al., 2016), with a poison model and 1000 bootstrap replications. FveIAA protein sequences from Arabidopsis (Dreher et al., 2006) were obtained from Phytozome (Goodstein et al., 2012).

The strawberry (F. vesca f. semperflorens) plants were procured from Xi’an University, Xi’an, China. The plants were grown in a greenhouse at a temperature of 20°C–25°C with a 14-h light/10-h dark cycle and relative humidity of 70%–85%. During the first week after anthesis, more than 100 small green (SG) fruits on 50 strawberry plants were labeled. Fruits were collected on days 7, 14, 25 days post fertilization, at three different stages: SG, BG (Big green), and R (Red), respectively. At each stage, sampling of uniformly sized fruits (N = 10) was done in triplicates. Small cubes of the receptacle (pulp) (0.5–0.8 cm³) were flash-frozen in liquid nitrogen and stored at −80°C. The other tissues were collected from healthy strawberry plants (N = 10) at the flowering stage and analyzed in triplicates. The newly growing and fully expanded leaves were used for the IAA treatments. The branch containing three-piece leaves were cut from the strawberry plants and dipped in 10 μM IAA (pH 6.6) solution. The branches treated with H₂O served as the control. The samples were collected 0, 1, 6, and 12 h after treatment. For each treatment, 24 branches were sampled from 12 different plants. All samples were flash-frozen in liquid nitrogen and stored at −80°C for RNA extraction.

TRIZOL Reagent was used to isolate total RNAs from the frozen samples (100–200 mg), followed by treatment with Turbo DNA-free TM kit to eliminate DNA contamination. After reverse transcription into cDNA, qRT-PCR was performed with the reaction mixture (20 μL) containing 1 μL forward/reverse specific primer (10 μM), 1 μL cDNA (30 ng/μL), and 10 μL SYBR Green Master Mix on an ABI Quant Studio tm 6 Flex Real-Time PCR System. The cycle parameters included 95°C for 3 min; 40 cycles at 95°C for 20 s, 58°C for 30 s, and 72°C for 30 s; 71 cycles increasing from 60°C to 95°C at 0.5°C per cycle for 30 s. FvActin (GenBank accession no. AB116565.1) was used as the internal reference to calculate the relative fold differences, and the data were analyzed using a previously described comparative CT method (2^−ΔΔCt) (Su et al., 2015). A P-value < 0.05 was regarded as statistically significant. NCBI primer blast was used to design the primers for qRT-PCR (Suppl. Tab. S1) using F. vesca mRNA as the reference database.

We identified and characterized 21 Aux/IAA genes in the F. vesca genome, which were labeled FveIAA1-21 based on their chromosomal localization. In-depth analysis of each predicted FveIAA, including chromosomal localization, gene length, deduced protein length, MW, pI, and exon numbers, was performed (Tab. 1). The FveIAA genes encode 181 (FveIAA16) to 370 amino acids (aa) (FveIAA3) with corresponding MWs of 20.45 to 39.84 kDa. The pI ranged from 5.23 (FveIAA5) to 8.61 (FveIAA10), indicating that different Aux/IAA proteins might function under different pH conditions. Sixteen FveIAAs were located in the nucleus, except FveIAA10, FveIAA11, FveIAA16, and FveIAA18, which were located in the chloroplasts; FveIAA12 was located in the mitochondria (Suppl. Tab. S2).

The 21 identified FveIAA genes were distributed across five chromosomes, mainly on chromosomes 1, 2, 4, 5, and 6, but were not found on chromosomes 3 and 7 (Fig. 1). These genes did not show random distribution on the chromosome due to gene clusters and hot regions. The unequal distribution of FveIAA genes suggested genetic variation during the evolutionary process (Li et al., 2020a, Li et al., 2019; Li et al., 2015). Thus, the segmental duplication and tandem duplication events were investigated to explore potential gene duplication within the strawberry genome. Four duplicated gene pairs (FveIAA2 and FveIAA4, FveIAA3 and FveIAA15, FveIAA7 and FveIAA11, FveIAA8 and FveIAA20) of FveIAAs, all occurring on different chromosomes with a possibility of segmental duplication, were observed in F. vesca (Fig. 2A). We hypothesized that apart from expanding the FveIAA gene family size, these segmental gene duplications also increased their functional diversity. We constructed a comparative syntenic map of F. vesca and Arabidopsis to further explore the phylogenetic mechanism of the F. vesca IAA gene family. A syntenic relationship was discovered between four FveIAA genes and Arabidopsis (Fig. 2B), indicating the importance of these genes from the Aux/IAA gene family during the evolutionary process.

Protein motif analysis by PFAM and sequence alignment revealed that 20 FveIAAs had four characteristic conserved domains: I–III and IX (Fig. 3).The domain I of most FveIAA proteins had a highly conserved LXLXLX motif, which served as a protein transcriptional repressor except FveIAA12, which was a non-canonical Aux/IAA protein missing domain I, also found in tomato and Medicago truncatula, indicating that these proteins may play specialized roles while mediating auxin response during plant growth and development. Most FveIAA proteins exhibited two distinct types of nuclear localization signals (NLS): A typical NLS (at the end of domain IV) and a bipartite NLS (between domains I and II).

A phylogenetic NJ tree was built based on the complete sequence alignment of 21 FveIAAs and 29 AtIAAs to explore the phylogenetic relationship between the Aux/IAA proteins of F. vesca and A. thaliana (Suppl. Tabs. S3 and S4). Based on the phylogenetic distribution, IAA proteins were classified into nine major groups (labeled 1 to 9) with well-supported bootstrap values (Fig. 4). Also, we analyzed the identity of the predicted Aux/IAA protein sequences between Arabidopsis and strawberry predicted based on the orthologs between these two species. We found no organism-specific group from the tree. In the common clades, there was an unequal distribution of the IAAs from the two organisms. For instance, Group 4 contained one FveIAA and four AtIAAs. Also, there was an unequal distribution of the FveIAAs in the nine groups. Groups 1–6, 8, and 9 included twenty FveIAA members (largest), which were located in the nucleus and the chloroplast, while Group 7 contained one gene (FveIAA12), which was located in the mitochondria (Suppl. Tab. S2).

We performed phylogenic and conserved motif analysis of the FveIAAs to explore the relationship between motifs and evolution and to identify the conserved regions (Fig. 5A). Twenty-one FveIAAs containing 2–10 conserved motifs were detected (Fig. 5B and Suppl. Fig. S1). Motifs 1 and 2 were common in all 21 FveIAAs, indicating that they were essential for basic functions of FveIAAs, while the other eight motifs were more or less missing from the FveIAAs. structure analysis showed that the gene structure of most of the FveIAA genes was identical to the AtIAAs, which included 3–5 exons and 2–4 introns, except for FveIAA15 (six exons and five introns) and FveIAA5 and FveIAA16 (each containing two exons and one intron) (Fig. 5C). Exon-intron structure analysis provides critical insights into the process of evolution of gene families. Motif composition, arrangements, and gene structures were consistent with the phylogenetic tree (Fig. 5).

The interaction between cis-elements and the corresponding trans-acting factors is known to promote gene regulation. PlantCARE was used to determine the probable cis-regulatory elements within the promoter region of the FveIAA genes to understand possible regulatory patterns of the FveIAAs. We found that all 21 FveIAAs promoter sequences contained several light-responsive elements, indicating that FveIAAs played a critical role in strawberry morphogenesis. Additionally, we found hormonal response-related cis-regulatory elements, such as auxin, methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA), and gibberellins (GA), as well as stress-responsive elements, including anaerobic induction, defense, drought, and low temperature in the promoter region of most FveIAA genes (Fig. 6). Some of the FveIAA genes contained tissue-specific elements (endosperm, meristem, and seed-specific activation) and circadian control elements.

A complete interaction network of the FveIAA family and its interacting proteins were predicted using the STRING tool to investigate the functions of the FveIAAs. Seventeen FveIAAs were identified in the interaction network except FveIAA4, FveIAA5, FveIAA6, and FveIAA20 (Fig. 7). Protein-protein relationship analysis revealed that most FveIAAs interacted with each other indicating collaborative functioning, such as in FveIAA1, FveIAA13, and FveIAA19. Also, the direct interaction between FveIAAs and the nodal proteins suggested the possibility of occasional indirect interaction between the FveIAAs, such as XP_004293901.1, a common core protein, induced indirect interaction between FveIAA19, FveIAA1, FveIAA15, FveIAA17, and FveIAA7 (Fig. 7). Functional annotation revealed that most of the common interacting proteins in the PPI network were involved in auxin-related signaling pathways (Suppl. Tab. S5).

We investigated the spatial-specific expression pattern of the 21 FveIAAs in different organs, including stems, roots, flowers, leaves, and fruits, to investigate the physiological function of FveIAAs (Fig. 8). All the FveIAAs were detected in different organs, except for FveIAA21, whose expression was downregulated in all organs. All the FveIAAs showed tissue-specific expression patterns in F. vesca. Most FveIAAs showed high stem-specific mRNA expression compared with other organs, except FveIAA4, FveIAA5, FveIAA13, and FveIAA18 whose expression level were upregulated in the fruit; FveIAA2, FveIAA9, FveIAA10, and FveIAA17 which showed root-specific expressions; FveIAA3, FveIAA11, and FveIAA16 also showed flower-specific expression; FveIAA1, FveIAA19, and FveIAA14, which showed leaf-specific expression. Transcriptional analysis of the FveIAAs showed tissue-specific expression in F. versa, suggesting that FveIAA genes might play distinct roles in different organs during strawberry development.

We detected the expression pattern of FveIAAs in leaves at 1, 6, and 12 h of post-IAA treatment using qRT-PCR to test the responsiveness to exogenous auxin stimuli (Fig. 9). We found that except for FveIAA21, which was downregulated in all organs, all other genes were auxin-responsive. Exogenous auxin upregulated the expression of most FveIAA genes after 1 h and 6 h; however, the expression was restored to near pre-stress levels after 12 h in FveIAA2, FveIAA3, FveIAA5, FveIAA7, FveIAA8, FveIAA14, FveIAA16 and FveIAA18. Also, elevated expression of FveIAA4, FveIAA10, FveIAA13, FveIAA17 and FveIAA20 was observed at all time points, including 12 h after treatment. In contrast, the expression of FveIAA9, FveIAA11, and FveIAA15 were downregulated by auxin at every time point. Thus, the complexity of auxin-regulated gene expression was reflected in the diverse pattern of expression of the FveIAA genes post-treatment with auxin.