Genome-Wide Gene Network Uncover Temporal and Spatial Changes of Genes in Auxin Homeostasis During Fruit Development in Strawberry (F. ×ananassa)

Genome-Wide Gene Network Uncover Temporal and Spatial Changes of Genes in Auxin Homeostasis During Fruit Development in Strawberry (F. ×ananassa) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-Wide Gene Network Uncover Temporal and Spatial Changes of Genes in Auxin Homeostasis During Fruit Development in Strawberry (F. ×ananassa) Yoon Jeong Jang, Taehoon Kim, Makou Lin, Jeongim Kim, Kevin Begcy, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4589609/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Sep, 2024 Read the published version in BMC Plant Biology → Version 1 posted 10 You are reading this latest preprint version Abstract Background The plant hormone auxin plays a crucial role in regulating important functions in strawberry fruit development. Although a few studies have described the complex auxin biosynthetic and signaling pathway in wild diploid strawberry ( Fragaria vesca ), the molecular mechanisms underlying auxin biosynthesis and crosstalk in octoploid strawberry fruit development are not fully characterized. To address this knowledge gap, comprehensive transcriptomic analyses were conducted at different stages of fruit development and compared between the achene and receptacle to identify developmentally regulated auxin biosynthetic genes and transcription factors during the fruit ripening process. Similar to wild diploid strawberry, octoploid strawberry accumulates high levels of auxin in achene compared to receptacle. Results Genes involved in auxin biosynthesis and conjugation, such as Tryptophan Aminotransferase of Arabidopsis (TAAs), YUCCA (YUCs), and Gretchen Hagen 3 (GH3s), were found to be primarily expressed in the achene, with low expression in the receptacle. Interestingly, several genes involved in auxin transport and signaling like Pin-Formed (PINs), Auxin/Indole-3-Acetic Acid Proteins (Aux/IAAs), Transport Inhibitor Response 1 / Auxin-Signaling F-Box (TIR/AFBs) and Auxin Response Factor (ARFs) were more abundantly expressed in the receptacle. Moreover, by examining DEGs and their transcriptional profiles across all six developmental stages, we identified key auxin-related genes co-clustered with transcription factors from the NAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY), Basic Region/ Leucine Zipper motif (bZIP), and APETALA2/Ethylene Responsive Factor (AP2/ERF) groups. Conclusions These results elucidate the complex regulatory network of auxin biosynthesis and its intricate crosstalk within the achene and receptacle, enriching our understanding of fruit development in octoploid strawberries. Auxin Strawberry fruit Plant hormone Achene Receptacle Transcriptomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The garden strawberry ( Fragaria × ananassa ), an allo-octoploid fruit crop (2n = 8x = 56), originated from the hybridization of two wild octoploid species, F. chiloensis subsp. chiloensis and F. virginiana subsp. Virginiana . It presents a genomic complexity with four subgenomes derived from different diploid progenitors: F. vesca , F. iinumae , F. nipponica , and F. viridis , as proposed by previous studies, [ 1 , 2 ]. Strawberries belonging to the Rosaceae family, are distinguished by their unique achenetum fruit architecture, where a single flower produces many achenes (fertilized ovaries), contrasting with the fleshy fruits of apples, peaches, and pears [ 3 , 4 ]. This distinction in fruit type, notably in strawberries and raspberries as aggregate fruits significantly influences morphology, production, storage, and distribution of their unique fruits [ 3 – 5 ]. Fertilization-induced fruit development in strawberries is of interest due to their unique flower and fruit structure. The strawberry fruit consists of numerous individual achenes embedded in a fleshy receptacle [ 6 ]. It is worth noting that what is commonly referred to as the fleshy fruit of a strawberry is actually a pseudocarp derived from the enlarged receptacle, while the true fruit (achene) is located on the epidermal layer [ 7 , 8 ]. The fruit set is the primary and crucial point for fruit growth in plants, and typically triggered by positive signals generated during the process of fertilization [ 9 ]. Fruit set can arise from different parts of the flower, such as the ovary in tomato ( Solanum lycopersicum ), receptacle in strawberry, and accessory part of hypanthium in apple ( Malus domestica ) [ 10 – 13 ]. In many plant species, the process of fruit set is largely dependent on the occurrence of fertilization. After successful pollination and fertilization, a series of physiological and molecular changes occur, ultimately leading to fruit development [ 14 ]. Flowers that do not undergo pollination and fertilization typically wither and fall off. Plant growth hormones such as auxin, gibberellin, abscisic acid (ABA), and cytokinin, are essential regulators of strawberry fruit development. These hormones regulate diverse biological processes, including promoting growth, increasing fruit size, and enhancing fruit set and yield [ 15 – 17 ]. Particularly, auxin is essential for fruit development, as it regulates cell division and differentiation in the receptacle, and its levels are dramatically regulated during fruit growth and ripening [ 18 ]. Although auxins such as indole-3-acetic acid (IAA) and phenylacetic acid (PAA) are crucial for plant growth and development, the complex biosynthetic pathways and enzyme redundancy within these pathways have hindered their complete understanding in plants, ranging from the model plant Arabidopsis to the crop model for fruit development [ 19 , 20 ]. Previous studies have demonstrated that auxin transport in the achene ceased during the late stages of mid-green fruit development to the ripening process of strawberry. This leads to a decrease in auxin levels in the receptacle and the subsequent ripening process [ 21 ]. Furthermore, prior studies also reported that removal of the achene from the receptacle after pollination inhibits fruit enlargement, while exogenous application of auxin promotes receptacle growth in the absence of achene [ 22 – 24 ]. Consequently, the achenes play a key role in auxin production needed to accelerate receptacle development [ 23 ]. Despite numerous studies on the complex biosynthesis and signaling pathways of auxin in wild diploid strawberries ( F . vesca ), the fundamental molecular mechanisms of auxin biosynthesis in octoploid strawberries, based on the high-quality haplotype-phased reference genome, have not yet been elucidated. Additionally, the intricacies of the differential gene expressions between achenes and the receptacle during fruit development in octoploid strawberries still need to be characterized. In Arabidopsis, IAA biosynthesis occurs mainly through the two-step biosynthetic pathways catalyzed by the amino transferases (Tryptophan aminotransferase of Arabidopsis (TAA) and TAA-related (TAR)), and monooxygenases belonging to the YUCCA (YUC) family that respectively convert Trp to Indole-3-pyruvic acid (IPyA) and IPyA to IAA [ 20 , 25 ]. Homologs of TAA/TAR and YUC have been identified in strawberry and implicated in various developmental processes, including fruit development. Several transcriptome profiling studies with F. vesca have shown that the auxin biosynthetic genes such as FvYUC and FvTAA , are predominantly induced in the endosperm after fertilization. Specifically, FvYUC5 , FvYUC11 , and FvTAR1 were primarily expressed in the achene, with low expression in the receptacle, and highly expressed in ghost (seedcoat + endosperm) and less so in the embryo and ovary wall [ 11 ]. It was also reported that FvYUC4 and Fv TAR2 were more abundantly expressed in the embryo, indicating their potential roles in embryonic development, while FvYUC10 , FvGA20ox1 , FvGA20ox2 , and FvGA3ox1 were present across various fruit tissues, exhibiting minimal to no expression within embryos. The expression of most of these biosynthetic genes gradually increases from stage 1 (open flower) to stages 5 (big green), likely due to the effect of fertilization [ 11 ]. Additionally, it was discovered that the wild strawberry F. vesca possesses nine genetic loci of YUCs genes, eight of which encode functional proteins. Specifically, the overexpression of FvYUC6 exhibited delayed flowering and male sterility in transgenic strawberry plants. Plants with reduced expression of FvYUC6 through RNAi demonstrated alterations in floral organ structure and root development [ 26 ]. In the transcriptome analysis of cultivated strawberries, two YUC genes, FaYUC1 and FaYUC2 (homologous to AtYUC6 and AtYUC4 , respectively), were identified. FaYUC1 and FaYUC2 are highly expressed in the large green fruit stage. Notably, the expression level of FaYUC2 is much higher compared to FaYUC1 [ 27 ]. During late-stage fruit development, FaYUC2 , FaTAR2 , and FaTAA1 were highly expressed in fruit receptacle [ 18 , 28 ]. Research on the plant hormone auxin, especially its perception and transcriptional regulation, is a critical aspect of plant phytohormone studies. Previous studies have identified auxin receptors, including the F-box protein Transport Inhibitor Response 1 (TIR1), which facilitates the degradation of Auxin/Indole-3-Acetic Acid (Aux/IAA) transcriptional repressors [ 29 ]. At low auxin levels, Aux/IAA proteins act as repressors for expressing target genes like Auxin Response Factors (ARF). However, when auxin levels increase, Aux/IAA proteins are degraded by the 26S proteasome, leading to release ARFs to activate auxin responses [ 30 ]. In F . vesca , studies on Aux/IAA and ARF genes suggest that the perception and transcriptional regulation of auxin involve 21 FvAux/IAA s and 19 FvARF s genes [ 11 ]. Furthermore, the expression of 19 FaAux/IAA genes was detected in the receptacle of cultivated strawberries. Most of these genes exhibited a consistent decrease in expression as the fruit transitioned from green to red stages. However, FaAux/IAA14b and FaAux/IAA11 showed maximum expression during the turning red stage, followed by a decrease in the red stage. For FaARF genes, while most showed low levels of expression from green to red stages, FaARF6a was notably more highly expressed during this transition compared to other FaARF genes [ 28 ]. Despite the large amount of evidence, a detailed molecular mechanism controlling auxin perception and gene regulation in octoploid strawberries, particularly within specific tissues such as achenes and receptacles remains unclear. In this study, a genome-wide transcriptome analysis was performed with a recent complete haplotype-phased octoploid strawberry reference genome of ‘Royal Royce’ (FaRR1) at various fruit developmental stages. Our results showed specific genes differentially expressed in receptacle and achenes during fruit development in octoploid strawberry. This deep transcriptome profiling identified genes involved in auxin biosynthetic signaling and metabolism pathways in strawberry. Moreover, our study identified novel transcription factors intricately linked to the auxin signaling network, marking a significant advancement in understanding the complex interplay between achenes and receptacles during the fruit development phase. RESULTS Accumulation of IAA in achene and receptacle during fruit development To investigate the developmental variations in IAA content of achenes and receptacles during fruit maturation, fruits of six different developmental stages were characterized. Stage 1; Small Green (SG), Stage 2; Medium Green (MG), Stage 3; Large Green (LG), Stage 4; White (W), Stage 5; Turning Red (TR), and Stage 6; Red (R) (Fig. 1 A). For each developmental stage, achenes and receptacles of individual fruits were separated and the concentration of IAA was quantified. The IAA content was consistently higher in achenes of all six stages, compared to the IAA detected in receptacles (Fig. 1 B and B). The highest IAA content was observed in the achenes of the SG and MG stage (approximately 4,000–7,000 pmol/g FW). The concentration of IAA in achenes decreased as the fruit matured and showed the lowest level of IAA at stage R (approximately 1000–2000 pmol/g FW) (Fig. 1 B). Based on one-way ANOVA Tukey’s test, achenes at stages SG and MG contained significantly greater IAA content compared to the R stage (P < 0.05). In contrast, receptacles exhibited significantly reduced IAA content in all stages of fruit development, but the highest IAA level was seen in the SG stage (approximately 50–110 pmol/g FW). A rapid decrease in IAA content at the MG stage of receptacles was observed, with concentrations dropping below 20 pmol/g FW (Fig. 1 C). This reduction in IAA content in the receptacles after the SG stage persisted to maturity at the R stage. Comparative transcriptome profiling analysis between achene and receptacle during fruit development To investigate the transcriptional changes during achene and receptacle development (SG, MG, LG, W, TR, and R) related to auxin homeostasis an RNA sequencing approach was used. Sequencing reads were mapped to the octoploid strawberry reference genome, cv. 'Royal Royce' (FaRR1) ( www.rosaceae.org ), resulting in a high average mapping rate of 95.6% for achene samples and 95.3% for receptacle samples (Table S1 and S2). Principal component analysis (PCA) was performed for the data quality assessment and exploratory analysis. Principal component 1 (PC1) accounted for 70% of the total variance, clearly delineating between achene and receptacle samples. Furthermore, principal component 2 (PC2) accounted for 22% of the total variance, distinguishing between the early and late stages of development. The PCA results suggest that the differences between achene and receptacle samples may reveal significant distinctions in the transcriptomes of these tissues across various stages of development (Supplemental Fig. 1.). To identify differentially expressed transcripts between achene and receptacle, we performed DESeq2 analysis using a significance cut-off padj < 0.05. The larger number of DEGs was identified in the comparison between achene and receptacle at the MG stage (32,686 DEGs total; 17,608 up-regulated and 15,078 down-regulated). In contrast, the comparison between achene and receptacle at the R stage yielded the smallest number of DEGs, totaling 7,131 DEGs from which 6,217 were up-regulated and 914 were down-regulated (Fig. 2 A, Supplemental Fig. 2 and Supplemental Table 3). Venn diagrams were generated for pairwise comparisons between achene and receptacle at each stage (SG, MG, LG, W, TR, and R), and a total of 2,892 genes were found to be expressed in all six developmental stages (Fig. 2 A). Moreover, stage-specific analyses revealed that 3,612, 4,396, 3,069, 1,583, 817, and 189 genes were uniquely expressed at SG, MG, LG, W, TR and R stages, respectively (Fig. 2 A). Intriguingly, our findings reveal a notable decrease in the number of differentially expressed genes, particularly during the later developmental stages, including the W, TR, and R stages. Our results also showed a significant increase in the expression levels of achene-specific transcripts across the different fruit developmental stages. Additionally, the expression patterns of achene or receptacle-specific genes varied throughout different developmental stages (Fig. 2 B). These observations highlight the substantial expression of achene-specific transcripts during fruit maturation. The observed variation in differentially gene expression patterns between achenes and receptacles during fruit maturation can be attributed to their distinct tissue compositions. Achenes, as more complex organs consisting of the embryo, endosperm, and seed coat, naturally display greater complexity and a higher number of differentially expressed genes. Conversely, the receptacle, a simpler organ composed of cortex and pith, shows less variability in gene expression due to its more uniform tissue structure. Dynamic differential gene expression in achene and receptacle during fruit development To elucidate the functional dynamics of gene expression between achene and receptacle tissues across six developmental stages, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using the ShinyGO web tool. We used a total of 2,892 DEGs identified across all different developmental stages of achene and receptacle (padj < 0.05) and subjected them to GO analysis to determine their associated Biological Process (BP), Cell Components (CC), and Molecular Functions (MF) associated with each gene (Fig. 3 and Supplemental Table 4). Within the BP category, DEGs between achene and receptacle common to each of the six developmental stages were primarily involved in pathways including the phenylpropanoid metabolic process, post-embryonic development, phenylpropanoid biosynthesis, as well as lignin metabolism and biosynthesis. Of particular note, we observed a common expression pattern of genes related to the response to hormone (GO:0009725) across all six stages (Fig. 3 , Supplemental Table 4). Additionally, in the CC category, the DEGs were found to relate to cellular components such as the vacuole, bounding membrane of organelle, vacuolar membrane, plant-type vacuole, and Golgi apparatus, among others (Fig. 3 , Supplemental Table 4). Interestingly, in the MF category, a significant enrichment of transcription factors and DNA binding activities was observed, including DNA-binding transcription factor activity, transcription regulator activity, transcription cis-regulatory region binding, transcription regulatory region nucleic acid binding, and sequence-specific DNA binding (Fig. 3 , Table S4 ). Taken together, our findings suggest a conservation of transcriptional regulation mechanisms in the DEGs of achene and receptacle in octoploid strawberry. We also performed a KEGG pathway enrichment analysis on the 2,892 overlapped stage candidate DEGs identified. The primary signaling pathways associated with strawberry in the comparison between achene and receptacle include: Biosynthesis of secondary metabolites (139), Phenylpropanoid biosynthesis (31), Plant hormone signal transduction (32), Cutin, suberine and wax biosynthesis (9), stilbenoid, diarylheptanoid, and gingerol biosynthesis (4), Diterpenoid biosynthesis (6), Fatty acid degradation (9), and Flavonoid biosynthesis (6) (Fig. 3 B and Supplemental Table 5). Identification of genes involved in auxin homeostasis in achene and receptacle during fruit development of octoploid strawberry The expression levels of genes associated with auxin homeostasis were determined in achene and receptacle, and a total of 164 genes were identified (Fig. 4 , Supplemental Table 6). The TAAs and YUCs are essential components for the biosynthesis of the major natural auxin in plants, IAA. As the IPyA pathway mediated by TAAs and YUCs produces the majority of free IAA, the expression of TAAs and YUCs is indispensable for various essential developmental processes, including fruit development. In the octoploid strawberry ‘Royal Royce’, we identified TAA1 homoeologous gene copies located in the four subgenome of chromosome 4, Fxa4Ag102498, Fxa4Bg102453, Fxa4Cg202161 , and Fxa4Dg101990 (Fig. 4 ). Among these copies, we found that Fxa4Bg102453, Fxa4Cg202161 , and Fxa4Dg101990 had high expression levels at the SG stage of achenes. Moreover, we identified homoeologous gene copies of TAR1/2 , including Fxa5Ag200524 , Fxa5Bg100503 , Fxa5Cg200481 , and Fxa5Dg200494 , respectively (Fig. 4 ). All four copies showed high expression levels only during the SG and MG stages. For TAR3/4 , four homoeologous gene copies, Fxa2Ag101303 , Fxa2Bg201139 , Fxa2Cg202166 , and Fxa2Dg201128 were identified. Interestingly, only both Fxa2Cg202166 and Fxa2Dg201128 were highly expressed in the receptacle, particularly during the MG stage, compared to the achene (Fig. 4 ). In addition, four FaYUC genes, FaYUC2 , FaYUC4 , FaYUC10 and FaYUC11 , were found in the octoploid strawberry. Protein analysis using the FvYUC2 sequence from diploid strawberry showed high similarity with four homoeologous copies of FaYUC2 , Fxa1Ag100425, Fxa1Bg200419, Fxa1Cg100379 , and Fxa1Dg200387 (Fig. 4 ). The gene expression result showed that only Fxa1Cg100379 appears to be dominantly expressed at the LG stage at achene, while the expression of the other three copies is either absent or very low. For FaYUC4 , interestingly, all four homoeologous copies of Fxa2Ag102968, Fxa2Bg202798, Fxa2Cg201097 , and Fxa2Dg202579 located in chromosome 2 are highly expressed during the MG stage of achene development. However, the expression of FaYUC4 was only found at the subgenome A ( Fxa2Ag102968 ), and B ( Fxa2Bg202798 ) at the SG and LG stages. The gene FaYUC10 has four homoeologous copies, Fxa2Ag102487 , Fxa2Bg202327 , Fxa2Cg203233 and Fxa2Dg202176 , in all subgenomes of chromosome 2. The expression levels were constitutively higher during the SG, MG, and LG stages of the achene compared to other stages. In receptacle, Fxa2Ag102487 , Fxa2Bg202327 , and Fxa2Cg203233 show relatively higher expression levels at the SG and MG stages. For FaYUC11 , three homoeologous gene copies, Fxa4Ag101793 , Fxa4Bg101727 , and Fxa4Cg201512 , were found and showed higher expression in all developmental stages of the achene compared to the receptacle (Fig. 4 ). Furthermore, we identified high expressions of GH3 genes, which belong to one of the major auxin-responsive gene families. FaGH3.1 , FaGH3.5 , FaGH3.6a , FaGH3.6b , FaGH3.9 , and FaGH3.17 showed expression levels with LogFC values from 0.48 to 6.06 (average = 2.4) at the SG stage and from 0.57 to 7.97 (average = 2.67) at the MG stage in achenes, respectively (Fig. 4 , Supplemental Tables 3 and 6). It has been known that GH3s play crucial roles in auxin homeostasis through conjugating free auxin with amino acids [ 31 ]. We identified that the FaGH3.5 homoeologous gene copies, Fxa6Ag104259 , Fxa6Bg103912 , Fxa6Cg103796 , and Fxa6Dg103714 , were highly expressed in the achene during the SG and MG stages. Interestingly, only one copy of FaGH3.6 ( Fxa3Ag100357 ) was highly expressed in receptacle during the SG and MG stages compared to the achene. Furthermore, FaGH3.6a , FaGH3.6b , FaGH3.9 , and FaGH3.17 showed predominantly high expression levels at the SG and MG stages of achene. Particularly, the three homoeologous copies of FaGH3.6b , Fxa3Bg201817 , Fxa3Cg101795 , and Fxa3Dg201745 , showed high expression levels in the achene at the SG stage (Fig. 4 ). For the PIN family genes, six homologs of PIN 1, 2, 4, 5, 8, and 10 were identified in F. × ananassa utilizing F. vesca as a query. Among them, FaPIN1 , FaPIN4 , FaPIN5 , and FaPIN8 exhibited stronger expression in the receptacle at SG than in achene at SG, but FaPIN10 showed the opposite pattern. Homologs of AUX/LAX genes, including AUX/LAX2 , AUX/LAX3 , and AUX/LAX4 , were identified in F. × ananassa . While FaAUX/LAX2 exhibited high gene expression at MG of achene. FaAUX/LAX4 showed stronger gene expression at SG and MG of achene compared to receptacle (Fig. 4 ). We also discovered nine Aux/IAA genes and 14 ARF gene family homologs in F. × ananassa . Among them, FaAux/IAA26b displayed differences in gene expression between achene and receptacle at MG. FaAux/IAA27a and FaAux/IAA27b showed slight differences in expression between achene and receptacle at SG and MG. Notably, at SG and MG, FaARF3 , FaARF6a , FaARF19a , and FaARF19b gene families exhibited higher expression in receptacle than in achene. However, FaARF1b displayed higher expression in achene from MG to W, and some genes including FaARF16a , FaARF16b , and FaARF17a,c showed higher expression in SG of achene compared to receptacle. Lastly, TIR1, AFB2, and AFB5 homologs were also identified in F. × ananassa . These auxin biosynthesis-related genes influence various aspects of F. × ananassa fruit effects, including growth, perception of auxin, and more, throughout the stages from early to late, suggesting their multifaceted impact on the fruit development of octoploid strawberry (Fig. 4 ). Transcription factors involved in auxin biosynthesis signaling networks between achenes and receptacles during fruit development To further understand auxin biosynthesis and transport between achene and receptacle, we used the entire list of DEG and clustered them across all six developmental stages based on their transcriptional expression. We identified four distinctive patterns of expression (Fig. 5 , Table S7 ). Cluster 1 was formed by 1209 genes with increasing expression in achene having the highest expression at LG. Genes in cluster 1 have a low or no expression in the six receptacle developmental stages (Fig. 5 A). Cluster 2 formed by 1229 genes showed a decreased expression from achene to receptacle in a developmental manner. The more advanced the developmental stage the lower the expression (Fig. 5 A). Cluster 3 showed an opposite pattern as cluster 1, having lower expression in the achene and higher expression in the receptacle (Fig. 5 A). Finally, cluster 4 formed by 177 genes, showed variable expression across developmental stages in achene and receptacles samples (Fig. 5 A). Since cluster 1 showed opposite developmental expression between achene and receptacle, we selected the entire set of genes identified in this cluster to conduct a correlation analysis to pinpoint transcription factors with shared expression profiles with genes involved in auxin biosynthesis and transport between achene and receptacle. A large portion of the strawberry genes are described as uncharacterized or unknown. Therefore, we removed them from the analysis. After removal of unrooted genes, the remaining genes were clustered using an edge confidence of at least 0.7. Our analysis yielded six main hubs containing transcription factors associated with auxin related genes (Fig. 5 B). These transcription factor hubs were grouped into NAC/WYKR (blue), MYB (green), APETALA2/Ethylene Responsive Factor (purple), homeobox (orange), heat shock transcription factor (red) and bZIP (black). Since we were interested in auxin biosynthesis and transport, we identified the auxin related genes (yellow) that clustered together with other transcription factors. Three out of the six hubs contained auxin related genes and were clustered with the NAC/WYKR, bZIP and APETALA2/Ethylene Responsive Factor hubs (Fig. 5 B). FvYUC10 ( Fxa2Ag102487 ) and FaPIN10 ( Fxa4Ag100605 ) clustered together with the APETALA2/Ethylene Responsive Factor group. FvARF1a ( Fxa5Dg202383 ) and FvARF16a ( Fxa6Dg101291 ) co-expressed with the NAC/WYKR hub and FvARF11 ( Fxa5Dg200932 ) with the bZIP group of transcription factor. Taken together, our analysis identifies potential transcription factors involved in the regulation of auxin and transport between achene and receptacles. Discussion The plant hormone auxin plays a fundamental role in regulating cell expansion and cell division [32, 33]. Previous studies indicate that fruit development is intricately linked to achene growth, with the maturation of fleshy fruits being inhibited in the absence of achene. This repression is relieved when fertilized achenes release auxin signals to the ovary wall and receptacle [34]. In strawberries, achenes are considered the principal sites for auxin accumulation, playing a pivotal role in regulating auxin distribution across the entire fruit, thereby influencing its development. [11, 21, 35]. Studies on auxin in wild diploid strawberry, Fragaria vesca , have shown that auxin increases both the width and length of the fruit during the early stages of fruit development, while gibberellic acid (GA), mainly promotes vertical growth [17]. Auxin Dynamics in Achene-Derived Auxin on Receptacle Growth and Strawberry Fruit Development Perkins-Veazie classified the ripening stages of strawberry fruit into four stages (green, white, pink, and red), with pink being reported as the turning point of ripening [8]. However, given that several previous studies have indicated the significant role of auxin synthesis and transport in the early developmental stages of strawberry growth, our research has not only divided the developmental process into six distinct stages, from early development to maturity but also separated achene and receptacle at each stage for hormone measurement and transcriptome analysis (Fig. 1). A previous study has reported a consistent reduction in IAA as the fruit transitions from the small green stage to the red stage, as shown in mixed tissue of achene and receptacle [36]. In this study, when analyzing the auxin content separately in the achene and receptacle of octoploid strawberries, we observed a significantly higher concentration of auxin in the achene compared to the receptacle, as observed in Fig. 1. This suggests that receptacle enlargement is likely due to auxin moving from achenes. Our results corroborate those of Estrada-Johnson et al. (2017) and Gu et al. (2019), who observed elevated IAA levels in achenes compared to receptacles during fruit development. Our study, however, distinguishes itself by employing a high-quality reference genome to perform detailed tissue-specific analyses across six developmental stages in octoploid strawberries, whereas Estrada-Johnson et al. (2017) focused on four stages. Moreover, while Gu et al. (2019) examined the transcriptome and auxin-related genes in diploid strawberries, our analysis extends to octoploid strawberry varieties, providing a more comprehensive understanding of auxin dynamics across different genomic complexities [28, 37]. One intriguing finding is the presence of approximately 50 - 110 pmol/g FW of IAA in the receptacles at the small green stage. This supports the result of temporal variations in auxin distribution, where auxin is synthesized within the achene and transported to the receptacle. Although present at a lower concentration compared to the achene, the IAA in the receptacle likely contributes to the coordination of fruit development, potentially regulating processes such as cell division and expansion. This supports the notion that even with a lower concentration, the receptacle serves as a receiver and transporter of IAA to modulate the growth and development of the strawberry fruit. Exploring Auxin Metabolism, Transport, Signaling, and Response Pathways in Achene and Receptacle To investigate the regulation of this phenomenon by specific genes, we conducted a genome-wide comprehensive analysis of the auxin-specific transcriptome in the achene and receptacle of octoploid strawberries using the recently developed high-quality haplotype-phased reference genome for octoploid strawberries [38]. We compared and analyzed the transcriptomes separately for the achene and receptacle at each developmental stage. According to previous research, strawberry fruit set is completed within two to four days after fertilization, and it has been shown that transcriptional regulation and signaling metabolic changes occur at fertilization. This pattern is consistent with our results (Fig. 1 and Fig. 4) [11]. To determine the collaborative action of gene families involved in the biosynthesis and transport of auxin, we conducted a DEG analysis using the criteria under padj 1 to identify significantly differentially expressed genes at each developmental stage. In this study, we examined the expressions of homologous genes previously studied, such as TAA, TAR, YUC, GH3, Aux/IAA, TIR/AFB, and ARF, and found that the genes related to auxin biosynthesis were highly expressed during SG or MG in the achene. Throughout the comprehensive analysis in this study, it was found that genes such as TAA1, YUC4, and GH3.6b play a pivotal role in auxin biosynthesis primarily within the achene. Additionally, the PIN gene family, essential for auxin efflux facilitation, markedly impacts auxin's directional flow owing to their unique intracellular localization [39, 40]. In octoploid strawberries, particularly during the SG stage, the expression of specific PIN genes ( FaPIN4 , FaPIN5 , and FaPIN8 ) is substantially elevated in the receptacle compared to the achene. This reaffirms the coordination of achene and receptacle in fruit TAA development. Previous studies have also reported the high expression of auxin-conjugating GH3 genes in ghost and ovary walls at the F. vesca [11]. Furthermore, the presence of IAA amide conjugates and highly abundant IAA-protein conjugates have been reported in the receptacle of strawberries (Archbold and Dennis 1984; Park, et al. 2006). One notable aspect to consider is that in many species, a multitude of Aux/IAA proteins modulate ARF-mediated transcription and offer extensive signaling interactions in various processes involving auxin [41]. In this research, we discovered that gene copies within the Aux/IAA family, specifically FaAux/IAA26a ( Fxa2Ag103448 , Fxa2Bg203233 , Fxa2Cg200618 , Fxa2Dg203024 ), FaAux/IAA27a (Fxa1Ag100497, Fxa1Bg200482 , Fxa1Cg100463, Fxa1Dg200444 ), and FaAux/IAA27b ( Fxa6Ag103081 , Fxa6Bg102848 , Fxa6Cg102729 , Fxa6Dg102648 ), alongside TIR/AFB genes such as FaTIR1 ( Fxa2Ag102496 , Fxa2Bg202336 , Fxa2Cg203243 , Fxa2Dg202184 ), FaAFB2 ( Fxa6Ag105135 ), and FaAFB5 ( Fxa5Ag203451 , Fxa5Bg103235 , Fxa5Cg202953 , Fxa5Dg203023 ), and ARF genes including FaARF3 ( Fxa3Dg20069 , Fxa3Cg100717 , Fxa3Ag100785 ), FaARF6a ( Fxa3Cg100416 , Fxa3Ag100462 ), FaARF19a ( Fxa1Cg100787 , Fxa1Dg200710 ), and FaARF19b ( Fxa4Ag100540 , Fxa4Bg100521 , Fxa4Cg200482 , Fxa4Dg100445 ), demonstrated notably higher expression levels in the receptacle than in the achene. This differential expression was particularly evident during the early developmental stages, from SG to LG, respectively. This intricate network of interactions may stem from gene expression driven by specific TIR/AFB, Aux/IAA, and ARF transcription factors in response to auxin [42]. Characterizing Auxin Transcription factors in Achene and Receptacle Our study specifically aime aimed to elucidate the transcription factors associated with the genes discovered during fruit development in octoploid strawberry. We focused on transcription factors that exhibit co-expression patterns with genes involved in auxin biosynthesis and transport within both achene and receptacle tissues. Our analysis revealed that several auxin-related genes were grouped alongside the NAC/WYKR, bZIP, and AP2/ERF transcription factor hubs. Specifically, FaYUC10 (Fxa2Ag102487) and FaPIN10 (Fxa4Ag100605) were associated with the AP2/ERF cluster. Auxin has been proposed to indirectly promote fruit ripening by stimulating the transcription of several ethylene components, leading to ethylene-induced fruit ripening and softening. Additionally, FaARF1a (Fxa5Dg202383) and FaARF16a (Fxa6Dg101291) demonstrated co-expression with the NAC/WYKR hub, while FaARF11 (Fxa5Dg200932) aligned with the bZIP transcription factor group. This collective data suggests potential transcription factors that may play a crucial role in regulating auxin biosynthesis and its transport dynamics between achene and receptacle during strawberry fruit ripening. Conclusions In our study provides comprehensive gene expression profiling specific at subgenome level of octoploid strawberries from early to late fruit developmental stages. This information highlights an important yet insufficiently explored area in the field of octoploid strawberry auxin research. Furthermore, identifying pattens of subgenome-specific gene expression would implicate pathways of auxin metabolites, as well as the transport and perception of auxin between achenes and receptacles (Fig. 6 ). Leveraging recently available high-quality haplotype-phased reference genomes and genome-wide transcriptome profiling analysis, we were able to unveil the network of genes in auxin homeostasis and enhance our understanding of the regulatory mechanisms during fruit development in strawberry. Materials and methods Sample preparations The samples of the strawberry fruits ( Fragaria × ananassa Duch. cv. Brilliance) were used for transcriptome analysis. The six different developmental stages of harvested fruits corresponded that stage 1 is Small Green (SG), stage 2 is Medium Green (MG), stage 3 is Large Green (LG), stage 4 is White (W), stage 5 is Turning Red (TR), and stage 6 is Red (R). In early January, the fruits were harvested from the UF strawberry field at Gulf Coast Research and Education Center in Balm, Florida. All stages of the fruits, including transcriptome sequencing biological replicated, were harvested simultaneously. Using the forceps and scalpels, each stage of the achenes and receptacle were separated from the fruits. Auxin measurement Approximately 20 mg (fresh weight) of achene and receptacle samples from 6 fruit stages were immediately frozen in liquid nitrogen and stored at -80°C. For IAA extraction and purification, frozen samples of achenes and receptacles were ground with pestles in liquid nitrogen and promptly submerged in 1.1 mL sodium phosphate buffer (50mM, pH 7.0) containing 0.1% diethyl dithiocarbamic acid sodium salt and 10 ng/mL of [ 13 C 6 ]-IAA as internal standard. Samples were incubated at 4°C with continuous shaking for 40 min and then centrifuged at 13000 g at 4°C for 15 min. After collecting supernatant and adjusting pH to 2.7, samples were purified by solid-phase extraction using Oasis™ HLB columns (WAT094225; Waters, MA, USA). The final elutes with 80% methanol were evaporated to dryness in vacuo and stored at -20°C until LC/MS analysis. The IAA detection method using liquid chromatography and mass spectrometer (LC-MS) was adapted from [ 43 ]. All samples were resuspended in MilliQ water and analyzed using Vanquish Horizon ultra-high performance liquid chromatography (UHPLC) installed with an Eclipse Plus C18 column (2.1 × 50 mm, 1.8 µm) (Agilent) and mass analysis was performed using a TSQ Altis Triple Quadrupole (Thermo Scientific) MS/MS system with an ion funnel. MRM parameters of the standards (precursor m/z, fragment m/z, radio frequency (RF) lens and collision energy) of each compound were optimized on the machine using direct infusion of the authentic standards. IAA and [ 13 C 6 ]-IAA were purchased from Cambridge Isotope Laboratories. For IAA detection, the mass spectrometer was operated in positive ionization mode at an ion spray voltage of 4800 V. Formic acid (0.1%) in water and 100% acetonitrile were used as mobile phases A and B, respectively, with a gradient program (0–95% solvent B over 4 min) at a flow rate of 0.4 mL per min. The sheath gas, aux gas and sweep gas were set at 50, 9 and 1 (arbitrary units), respectively. Ion transfer tube temperature and vaporizer temperature were set at 325°C and 350°C, respectively. For MRM monitoring, both Q1 and Q3 resolutions were set at 0.7 FWHM with collision-induced dissociation (CID) gas at 1.5 mTorr. The scan cycle time was 0.8 s. MRM for IAA was used to monitor parent ion → product ion reactions for each analyte as follows: m/z 175.983→130.071 (CE, 18 V) for IAA; m/z 182.091→136 (CE, 18 V) for [ 13 C 6 ]-IAA. IAA analysis was conducted with three biological replicates. Transcriptome data analysis The total RNA from separate achene and receptacle at six development stage with three replications per sample were extracted by the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, MO, USA) as fallowing the manufacturer protocol. To generate RNA-seq for illumina sequencing library were fallowed Illumina sequencing protocol. The resulting sequencing library were performed pair-end sequenced (2 x 150bp) by Illumina NovaSeq instruments at Novogene Bioinformatics Institute, Beijing, China. Raw read sequences obtained from 36 sequenced libraries were quality trimmed and filtered using Trimmomatic [ 44 ]. Data quality was assessed using FastQC. Trimmed paired end reads were aligned to the ‘Royal Royce’ octoploid genome [ 45 ] using Hisat2 [ 46 ]. Differentially expressed genes (DEG) analysis was conducted using the DESeq2 package in R script [ 47 ] using a fold change >|1| and P < 0.05 (after the false discovery rate adjustment for multiple testing (FDR) < 0.05) for the null hypothesis. Total number of reads mapped to each gene was used to calculate transcripts per million (TPM) values, which were determined using a custom Python script. Gene Ontology (GO) enrichment analysis was conducted using Arabidopsis gene information provided by the Royal Royce genome annotation database [ 45 ]. The analysis was conducted through the ShinyGO tool (version 0.77, http://bioinformatics.sdstate.edu/go/ , [ 48 ]), applying a P-value cutoff of ≤ 0.05 (FDR) and default options. Confirmation of homology of octoploid strawberry auxin hormone pathway genes Genes associated with auxin biosynthesis were explored in the octoploid strawberry involved utilizing known genes from F. vesca and previous literature. BlastP was performed with significant criteria under e-value = 0, pident 100 based on the protein sequence against the Royal Royce reference genome [ 38 , 49 ] for each hormone-related gene [ 50 ]. K-means Clustering and Gene Co-expression Analysis Transcripts per million (TPM) counts were used as input for the K-means clustering analysis. TPM values were averaged for the replicates of each tissue (achene or receptacle) at any given stage (Stage 1 to Stage 6). Averaged TPM values were normalized to log 2 (TPM + 1), scaled, and used as input for clustering analysis. DEGs were categorized into four clusters using the k-means algorithm implemented using the R programming language. In order to identify transcriptional correlations among genes with shared expression profile analysis achene or receptacle, the cor function was implemented in the R package WGCNA. Then, genes with shared expression profiles were considered as seed candidates and were used to build a gene co-expression analysis to obtain direct and indirect interactions. A high confidence score of 0.7 was used as a threshold [ 51 ]. Only high levels of confidence interactions were considered as valid as used in the final gene co-expression analysis. Abbreviations Abscisic Acid (ABA) Apetala2/Ethylene Responsive Factor (AP2/ERF) Auxin Response Factor (ARFs) Auxin/Indole-3-Acetic Acid Proteins (Aux/IAAs) Basic Region/ Leucine Zipper Motif (bZIP) Biological Process (BP) Cell Components (CC) Gene Ontology (GO) Gretchen Hagen 3 (GH3s) Indole-3-Acetic Acid (IAA) Indole-3-Pyruvic Acid (IPyA) Kyoto Encyclopedia Of Genes And Genomes (KEGG) Large Green (LG) Medium Green (MG) Molecular Functions (MF) NAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY), Phenylacetic Acid (PAA) Pin-Formed (PINs) Principal Component Analysis (PCA) Red (R) Royal Royce (FaRR1) Small Green (SG) Taa-Related (TAR) Transport Inhibitor Response 1 (TIR1) Transport Inhibitor Response 1 / Auxin-Signaling F-Box (TIR/AFBs) Tryptophan Aminotransferase Of Arabidopsis (TAA) Turning Red (TR) White (W) YUCCA (YUCs) Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials High-throughput sequencing data analyzed in present study are available under NCBI BioProject PRJNA1010111. The online version contains Supporting Information available Competing interests The authors declare that they have no competing interests Funding This research is supported by grants from the United States Department of Agriculture National Institute of Food and Agriculture (NIFA) Specialty Crops Research Initiative (SCRI) “Delivering Breeding and Management Solutions to Prevent Losses to Emerging and Expanding Disease Threats in Strawberry” under award number (#2022-51181-38328) to S.L and NSF-IOS-CAREER- 2142898 to J.K. Author contributions YJ, KB, JK and SL contributed to the study design and drafted the article. YJ, JK, TK, KB, HH, ML, ZL, VW, SL and analyzed the experiment results, prepared figures and tables. All authors read and approved the manuscript. Acknowledgements We extend our gratitude to Dr. Vance M. Whitaker for providing the fruit materials used in this study. 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Supplementary Files 01SupplementaryFigSfinal.pdf 02SupplementaryTableS1S2.pdf 03SupplementaryTableS3.xlsx 04SupplementaryTableS4.xlsx 05SupplementaryTableS5.xlsx 06SupplementaryTableS6.xlsx 07SupplementalTableS7.xlsx SupportingInformation.docx Cite Share Download PDF Status: Published Journal Publication published 20 Sep, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 08 Jul, 2024 Reviews received at journal 05 Jul, 2024 Reviews received at journal 01 Jul, 2024 Reviewers agreed at journal 20 Jun, 2024 Reviewers agreed at journal 19 Jun, 2024 Reviewers invited by journal 19 Jun, 2024 Editor invited by journal 19 Jun, 2024 Editor assigned by journal 19 Jun, 2024 Submission checks completed at journal 19 Jun, 2024 First submitted to journal 16 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4589609","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":321210451,"identity":"0165d2a9-0100-448b-81bf-09a7e0df87f2","order_by":0,"name":"Yoon Jeong Jang","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Yoon","middleName":"Jeong","lastName":"Jang","suffix":""},{"id":321210452,"identity":"ff7855e5-ceb5-45d6-83c5-2e1ca3ae68b0","order_by":1,"name":"Taehoon Kim","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Taehoon","middleName":"","lastName":"Kim","suffix":""},{"id":321210454,"identity":"9d0d7d8b-369d-4cef-b5ec-9ed5ec8c5285","order_by":2,"name":"Makou Lin","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Makou","middleName":"","lastName":"Lin","suffix":""},{"id":321210457,"identity":"39331deb-8071-4740-a4e6-85ecc59e81dd","order_by":3,"name":"Jeongim Kim","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Jeongim","middleName":"","lastName":"Kim","suffix":""},{"id":321210459,"identity":"4a41e6ac-ce0b-4fc7-a680-d219bacd5831","order_by":4,"name":"Kevin Begcy","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Begcy","suffix":""},{"id":321210461,"identity":"2dbc3fed-c5c9-4ac5-8c5b-6dfd0f9873f8","order_by":5,"name":"Zhongchi Liu","email":"","orcid":"","institution":"University of Maryland","correspondingAuthor":false,"prefix":"","firstName":"Zhongchi","middleName":"","lastName":"Liu","suffix":""},{"id":321210464,"identity":"21e36345-c9cf-4187-88b9-0ff107f74698","order_by":6,"name":"Seonghee Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYLACxgYGBjZm/ocPGGwsgNwEIrXwsfcwGzCkSZCgRY7nDJsEUVrk25uPPfy6w0aOTSL3WDVPggQDP3uOAV4tBmeOpRvLnkkzZpPIS7sN0iLZ84aAFokcM2nJtsOJbRIJZrd5f0gwGNwgYIv8/DcgLf/rQVqKQbbYE9LCcIPHTPJj24EENp4zZswgLUB7CfklLU2asS3ZsI29LVlyToIEj8SZZwX4HdZ++JjkzzY7eflm5oMf3iTYyPG3J2/A7zAgYOZB4vDgVIYMGH8QpWwUjIJRMApGLAAAsjFAOfIsUOYAAAAASUVORK5CYII=","orcid":"","institution":"University of Florida","correspondingAuthor":true,"prefix":"","firstName":"Seonghee","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2024-06-16 12:08:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4589609/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4589609/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-05577-5","type":"published","date":"2024-09-20T15:57:23+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":59693843,"identity":"c85bb3b6-93ee-47d7-a0d7-63837f3bece4","added_by":"auto","created_at":"2024-07-05 02:15:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":533615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIAA levels in strawberry achene and receptacle during fruit development.\u003c/strong\u003e (a) Attached and detached achene and receptacle developmental stages of cultivar ‘Florida Brilliance’ (\u003cem\u003eFragaria\u003c/em\u003e × \u003cem\u003eananassa\u003c/em\u003e Duch). Stage 1; Small Green (SG), Stage 2; Medium Green (MG), Stage 3; Large Green (LG), Stage 4; White (W), Stage 5; Turning Red (TR), and Stage 6; Red (R). Scale bar: 2cm. IAA contents in (b) achenes and (c) receptacles. Statistical analysis was conducted using one-way ANOVA Tukey’s test (P \u0026lt; 0.05). Different letters indicate significant differences in the amount of free IAA. Mean and standard deviation are shown (n=3).\u003c/p\u003e","description":"","filename":"FinalFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/38c66b454e9e46aa998f6c4e.png"},{"id":59693841,"identity":"d9d7953b-a137-4da4-b6d1-30a353334172","added_by":"auto","created_at":"2024-07-05 02:15:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":174823,"visible":true,"origin":"","legend":"\u003cp\u003eTranscriptional changes across six stages of fruit development in achene and receptacle tissues. (a) Venn diagram displaying unique and overlapping sets of 2,892 DEGs identified in achene and receptacle tissues, padj \u0026lt; 0.05. (b) Heatmap analysis of 2,892 overlapping DEGs. Gene expression values are shown as Z-score, padj \u0026lt; 0.05. Developmental stages are color coded. Small Green (SG) in red. Medium Green (MG) in yellow. Large Green (LG) in green. White (W) in purple. Turning Red (TR) in blue and Red (R) stage in black.\u003c/p\u003e","description":"","filename":"FinalFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/030428896422df49e82712d5.png"},{"id":59694230,"identity":"f66f7136-9741-4ac0-8c0b-28ac63e30e48","added_by":"auto","created_at":"2024-07-05 02:23:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180346,"visible":true,"origin":"","legend":"\u003cp\u003eGO and KEGG pathway enrichment analyses of differentially expressed genes in achene and receptacle. A) Fold enrichment for GO biological process (BP), cellular component (CC) and Molecular function (MF). The x-axis indicates the -log10 FDR, and the y-axis shows the GO terms. Colors represent the degree of enrichment. B) Bubble diagram highlights eight significant KEGG pathways, with circle sizes reflecting the number of DEG counts. Pathways with the highest significance (FDR \u0026lt; 0.05) are delineated within respective clusters.\u003c/p\u003e","description":"","filename":"FinalFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/51c9821d31fd6359a5ab7a46.png"},{"id":59693847,"identity":"4bffad33-640d-47c1-90ae-ee5f7be212e7","added_by":"auto","created_at":"2024-07-05 02:15:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":271402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptional analysis reveals differential gene expression patterns of auxin metabolism, transport, signaling, and response genes in achene and receptacle during fruit development. \u003c/strong\u003eGene expression levels were normalized to z-scores using a log2(1+TPM) transformation and visualized using a color scale heatmap plot. Color gradients ranging from green to red represent the respective gene expression values, with green indicating lower expression and red indicating higher expression. Small Green (SG), Medium Green (MG), Large Green (LG), White (W), Turning Red (TR) and Red (R) stages.\u003c/p\u003e","description":"","filename":"FinalFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/288928d944b68d8c868fd198.png"},{"id":59693851,"identity":"6a3b8de0-29ce-4c55-b7af-119350a669aa","added_by":"auto","created_at":"2024-07-05 02:15:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1337250,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNovel transcription factors involved in gene regulation between achenes and receptacles of strawberry fruits. \u003c/strong\u003e(a) Clustering analysis including all differentially expressed genes. (b) Gene network analysis of differentially expressed genes between achenes and receptacles. Transcription factors are shown in red. Auxin related genes are shown in yellow. All other differentially expressed genes are shown in green. A threshold of 0.7 of edge confidence was used. A detailed list of the genes included in the gene interaction analysis can be found in Supporting Table S7.\u003c/p\u003e","description":"","filename":"FinalFigures5.png","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/b03a985cb371c3faefd04fab.png"},{"id":59693853,"identity":"bb99ec31-387d-40d3-8969-667f4c27ea3c","added_by":"auto","created_at":"2024-07-05 02:15:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":418928,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of auxin-related genes transcriptional behavior in achene and receptable of strawberry fruits. \u003c/strong\u003eAuxin signaling pathway in strawberry development, highlighting key genetic components. Metabolic processes convert anthranilate to tryptophan, involving genes like TAA1 and TAT1,2, which lead to the production of the hormone auxin (IAA). The transport of auxin is facilitated by PIN and AUX/LAX proteins, crucial for establishing concentration gradients within the plant. In response to auxin levels, the Aux/IAA proteins may regulate gene expression by either repressing or permitting the activity of ARF proteins. High auxin levels trigger the degradation of Aux/IAA repressors, allowing ARF to activate transcription. Genes such as TIR1 and AFB2,5 are essential for auxin perception, initiating the proteasomal degradation pathway.\u003c/p\u003e","description":"","filename":"FinalFigures6.png","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/b844ceac268ccfa1697f056b.png"},{"id":65103965,"identity":"423bba90-5e43-489d-ae71-d616cec9679b","added_by":"auto","created_at":"2024-09-23 16:10:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5211692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/39c723d1-8c2d-4e83-be15-1ed58540f1ee.pdf"},{"id":59694231,"identity":"c1858604-36c4-4231-8ec7-bd0cc006ec9f","added_by":"auto","created_at":"2024-07-05 02:23:20","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":93957,"visible":true,"origin":"","legend":"","description":"","filename":"01SupplementaryFigSfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/9512d1bae31c8c38721e2467.pdf"},{"id":59694232,"identity":"22918da8-4ae1-44dd-9311-8d641d293334","added_by":"auto","created_at":"2024-07-05 02:23:20","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":111413,"visible":true,"origin":"","legend":"","description":"","filename":"02SupplementaryTableS1S2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/47e1a5de466fa4d3e27cce82.pdf"},{"id":59693854,"identity":"c2a5ae18-9d77-4a47-ad36-09c66d6ef054","added_by":"auto","created_at":"2024-07-05 02:15:21","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":18456626,"visible":true,"origin":"","legend":"","description":"","filename":"03SupplementaryTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/690281a76b55aa56c89fc3f2.xlsx"},{"id":59693849,"identity":"7fdc479a-0519-461e-8b82-a96fff8160b4","added_by":"auto","created_at":"2024-07-05 02:15:20","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":35135,"visible":true,"origin":"","legend":"","description":"","filename":"04SupplementaryTableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/07c4fd0c6f8dcb83222339df.xlsx"},{"id":59693848,"identity":"4117354c-1d16-4db9-b55a-69d11d196d5a","added_by":"auto","created_at":"2024-07-05 02:15:20","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":20390,"visible":true,"origin":"","legend":"","description":"","filename":"05SupplementaryTableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/04664ec45a2b815206db02d0.xlsx"},{"id":59694233,"identity":"9c4a629e-e8f7-4d70-86c5-dd8db12dfa99","added_by":"auto","created_at":"2024-07-05 02:23:20","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":42270,"visible":true,"origin":"","legend":"","description":"","filename":"06SupplementaryTableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/d47dac0b491b08e86e845b7f.xlsx"},{"id":59693852,"identity":"3c39d572-b4e9-45c9-8e96-c5aa33281aa1","added_by":"auto","created_at":"2024-07-05 02:15:20","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":96539,"visible":true,"origin":"","legend":"","description":"","filename":"07SupplementalTableS7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/7774cbcb9a05196ed05db3e1.xlsx"},{"id":59693845,"identity":"99f0dbbc-d60f-4ee7-85bb-0db6d0a9a308","added_by":"auto","created_at":"2024-07-05 02:15:20","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":14914,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-4589609/v1/2a5943d56a70e8cba7ca7746.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Wide Gene Network Uncover Temporal and Spatial Changes of Genes in Auxin Homeostasis During Fruit Development in Strawberry (F. ×ananassa)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe garden strawberry (\u003cem\u003eFragaria\u003c/em\u003e \u0026times;\u003cem\u003eananassa\u003c/em\u003e), an allo-octoploid fruit crop (2n\u0026thinsp;=\u0026thinsp;8x\u0026thinsp;=\u0026thinsp;56), originated from the hybridization of two wild octoploid species, \u003cem\u003eF. chiloensis\u003c/em\u003e subsp. \u003cem\u003echiloensis\u003c/em\u003e and \u003cem\u003eF. virginiana\u003c/em\u003e subsp. \u003cem\u003eVirginiana\u003c/em\u003e. It presents a genomic complexity with four subgenomes derived from different diploid progenitors: \u003cem\u003eF. vesca\u003c/em\u003e, \u003cem\u003eF. iinumae\u003c/em\u003e, \u003cem\u003eF. nipponica\u003c/em\u003e, and \u003cem\u003eF. viridis\u003c/em\u003e, as proposed by previous studies, [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Strawberries belonging to the Rosaceae family, are distinguished by their unique achenetum fruit architecture, where a single flower produces many achenes (fertilized ovaries), contrasting with the fleshy fruits of apples, peaches, and pears [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This distinction in fruit type, notably in strawberries and raspberries as aggregate fruits significantly influences morphology, production, storage, and distribution of their unique fruits [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Fertilization-induced fruit development in strawberries is of interest due to their unique flower and fruit structure. The strawberry fruit consists of numerous individual achenes embedded in a fleshy receptacle [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is worth noting that what is commonly referred to as the fleshy fruit of a strawberry is actually a pseudocarp derived from the enlarged receptacle, while the true fruit (achene) is located on the epidermal layer [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The fruit set is the primary and crucial point for fruit growth in plants, and typically triggered by positive signals generated during the process of fertilization [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Fruit set can arise from different parts of the flower, such as the ovary in tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e), receptacle in strawberry, and accessory part of hypanthium in apple (\u003cem\u003eMalus domestica\u003c/em\u003e) [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In many plant species, the process of fruit set is largely dependent on the occurrence of fertilization. After successful pollination and fertilization, a series of physiological and molecular changes occur, ultimately leading to fruit development [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Flowers that do not undergo pollination and fertilization typically wither and fall off.\u003c/p\u003e \u003cp\u003ePlant growth hormones such as auxin, gibberellin, abscisic acid (ABA), and cytokinin, are essential regulators of strawberry fruit development. These hormones regulate diverse biological processes, including promoting growth, increasing fruit size, and enhancing fruit set and yield [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Particularly, auxin is essential for fruit development, as it regulates cell division and differentiation in the receptacle, and its levels are dramatically regulated during fruit growth and ripening [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Although auxins such as indole-3-acetic acid (IAA) and phenylacetic acid (PAA) are crucial for plant growth and development, the complex biosynthetic pathways and enzyme redundancy within these pathways have hindered their complete understanding in plants, ranging from the model plant Arabidopsis to the crop model for fruit development [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Previous studies have demonstrated that auxin transport in the achene ceased during the late stages of mid-green fruit development to the ripening process of strawberry. This leads to a decrease in auxin levels in the receptacle and the subsequent ripening process [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, prior studies also reported that removal of the achene from the receptacle after pollination inhibits fruit enlargement, while exogenous application of auxin promotes receptacle growth in the absence of achene [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consequently, the achenes play a key role in auxin production needed to accelerate receptacle development [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Despite numerous studies on the complex biosynthesis and signaling pathways of auxin in wild diploid strawberries (\u003cem\u003eF\u003c/em\u003e. \u003cem\u003evesca\u003c/em\u003e), the fundamental molecular mechanisms of auxin biosynthesis in octoploid strawberries, based on the high-quality haplotype-phased reference genome, have not yet been elucidated. Additionally, the intricacies of the differential gene expressions between achenes and the receptacle during fruit development in octoploid strawberries still need to be characterized.\u003c/p\u003e \u003cp\u003eIn Arabidopsis, IAA biosynthesis occurs mainly through the two-step biosynthetic pathways catalyzed by the amino transferases (Tryptophan aminotransferase of Arabidopsis (TAA) and TAA-related (TAR)), and monooxygenases belonging to the YUCCA (YUC) family that respectively convert Trp to Indole-3-pyruvic acid (IPyA) and IPyA to IAA [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Homologs of TAA/TAR and YUC have been identified in strawberry and implicated in various developmental processes, including fruit development. Several transcriptome profiling studies with \u003cem\u003eF. vesca\u003c/em\u003e have shown that the auxin biosynthetic genes such as \u003cem\u003eFvYUC\u003c/em\u003e and \u003cem\u003eFvTAA\u003c/em\u003e, are predominantly induced in the endosperm after fertilization. Specifically, \u003cem\u003eFvYUC5\u003c/em\u003e, \u003cem\u003eFvYUC11\u003c/em\u003e, and \u003cem\u003eFvTAR1\u003c/em\u003e were primarily expressed in the achene, with low expression in the receptacle, and highly expressed in ghost (seedcoat\u0026thinsp;+\u0026thinsp;endosperm) and less so in the embryo and ovary wall [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It was also reported that \u003cem\u003eFvYUC4\u003c/em\u003e and Fv\u003cem\u003eTAR2\u003c/em\u003e were more abundantly expressed in the embryo, indicating their potential roles in embryonic development, while \u003cem\u003eFvYUC10\u003c/em\u003e, \u003cem\u003eFvGA20ox1\u003c/em\u003e, \u003cem\u003eFvGA20ox2\u003c/em\u003e, and \u003cem\u003eFvGA3ox1\u003c/em\u003e were present across various fruit tissues, exhibiting minimal to no expression within embryos. The expression of most of these biosynthetic genes gradually increases from stage 1 (open flower) to stages 5 (big green), likely due to the effect of fertilization [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, it was discovered that the wild strawberry \u003cem\u003eF. vesca\u003c/em\u003e possesses nine genetic loci of \u003cem\u003eYUCs\u003c/em\u003e genes, eight of which encode functional proteins. Specifically, the overexpression of \u003cem\u003eFvYUC6\u003c/em\u003e exhibited delayed flowering and male sterility in transgenic strawberry plants. Plants with reduced expression of \u003cem\u003eFvYUC6\u003c/em\u003e through RNAi demonstrated alterations in floral organ structure and root development [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the transcriptome analysis of cultivated strawberries, two \u003cem\u003eYUC\u003c/em\u003e genes, \u003cem\u003eFaYUC1\u003c/em\u003e and \u003cem\u003eFaYUC2\u003c/em\u003e (homologous to \u003cem\u003eAtYUC6\u003c/em\u003e and \u003cem\u003eAtYUC4\u003c/em\u003e, respectively), were identified. \u003cem\u003eFaYUC1\u003c/em\u003e and \u003cem\u003eFaYUC2\u003c/em\u003e are highly expressed in the large green fruit stage. Notably, the expression level of \u003cem\u003eFaYUC2\u003c/em\u003e is much higher compared to \u003cem\u003eFaYUC1\u003c/em\u003e [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. During late-stage fruit development, \u003cem\u003eFaYUC2\u003c/em\u003e, \u003cem\u003eFaTAR2\u003c/em\u003e, and \u003cem\u003eFaTAA1\u003c/em\u003e were highly expressed in fruit receptacle [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eResearch on the plant hormone auxin, especially its perception and transcriptional regulation, is a critical aspect of plant phytohormone studies. Previous studies have identified auxin receptors, including the F-box protein Transport Inhibitor Response 1 (TIR1), which facilitates the degradation of Auxin/Indole-3-Acetic Acid (Aux/IAA) transcriptional repressors [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. At low auxin levels, Aux/IAA proteins act as repressors for expressing target genes like Auxin Response Factors (ARF). However, when auxin levels increase, Aux/IAA proteins are degraded by the 26S proteasome, leading to release ARFs to activate auxin responses [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In \u003cem\u003eF\u003c/em\u003e. \u003cem\u003evesca\u003c/em\u003e, studies on Aux/IAA and ARF genes suggest that the perception and transcriptional regulation of auxin involve 21 \u003cem\u003eFvAux/IAA\u003c/em\u003es and 19 \u003cem\u003eFvARF\u003c/em\u003es genes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, the expression of 19 \u003cem\u003eFaAux/IAA\u003c/em\u003e genes was detected in the receptacle of cultivated strawberries. Most of these genes exhibited a consistent decrease in expression as the fruit transitioned from green to red stages. However, \u003cem\u003eFaAux/IAA14b\u003c/em\u003e and \u003cem\u003eFaAux/IAA11\u003c/em\u003e showed maximum expression during the turning red stage, followed by a decrease in the red stage. For \u003cem\u003eFaARF\u003c/em\u003e genes, while most showed low levels of expression from green to red stages, \u003cem\u003eFaARF6a\u003c/em\u003e was notably more highly expressed during this transition compared to other \u003cem\u003eFaARF\u003c/em\u003e genes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Despite the large amount of evidence, a detailed molecular mechanism controlling auxin perception and gene regulation in octoploid strawberries, particularly within specific tissues such as achenes and receptacles remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, a genome-wide transcriptome analysis was performed with a recent complete haplotype-phased octoploid strawberry reference genome of \u0026lsquo;Royal Royce\u0026rsquo; (FaRR1) at various fruit developmental stages. Our results showed specific genes differentially expressed in receptacle and achenes during fruit development in octoploid strawberry. This deep transcriptome profiling identified genes involved in auxin biosynthetic signaling and metabolism pathways in strawberry. Moreover, our study identified novel transcription factors intricately linked to the auxin signaling network, marking a significant advancement in understanding the complex interplay between achenes and receptacles during the fruit development phase.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAccumulation of IAA in achene and receptacle during fruit development\u003c/h2\u003e \u003cp\u003eTo investigate the developmental variations in IAA content of achenes and receptacles during fruit maturation, fruits of six different developmental stages were characterized. Stage 1; Small Green (SG), Stage 2; Medium Green (MG), Stage 3; Large Green (LG), Stage 4; White (W), Stage 5; Turning Red (TR), and Stage 6; Red (R) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). For each developmental stage, achenes and receptacles of individual fruits were separated and the concentration of IAA was quantified. The IAA content was consistently higher in achenes of all six stages, compared to the IAA detected in receptacles (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and B). The highest IAA content was observed in the achenes of the SG and MG stage (approximately 4,000\u0026ndash;7,000 pmol/g FW). The concentration of IAA in achenes decreased as the fruit matured and showed the lowest level of IAA at stage R (approximately 1000\u0026ndash;2000 pmol/g FW) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Based on one-way ANOVA Tukey\u0026rsquo;s test, achenes at stages SG and MG contained significantly greater IAA content compared to the R stage (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, receptacles exhibited significantly reduced IAA content in all stages of fruit development, but the highest IAA level was seen in the SG stage (approximately 50\u0026ndash;110 pmol/g FW). A rapid decrease in IAA content at the MG stage of receptacles was observed, with concentrations dropping below 20 pmol/g FW (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This reduction in IAA content in the receptacles after the SG stage persisted to maturity at the R stage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eComparative transcriptome profiling analysis between achene and receptacle during fruit development\u003c/h2\u003e \u003cp\u003eTo investigate the transcriptional changes during achene and receptacle development (SG, MG, LG, W, TR, and R) related to auxin homeostasis an RNA sequencing approach was used. Sequencing reads were mapped to the octoploid strawberry reference genome, cv. 'Royal Royce' (FaRR1) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.rosaceae.org\" target=\"_blank\"\u003ewww.rosaceae.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.rosaceae.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), resulting in a high average mapping rate of 95.6% for achene samples and 95.3% for receptacle samples (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). Principal component analysis (PCA) was performed for the data quality assessment and exploratory analysis. Principal component 1 (PC1) accounted for 70% of the total variance, clearly delineating between achene and receptacle samples. Furthermore, principal component 2 (PC2) accounted for 22% of the total variance, distinguishing between the early and late stages of development. The PCA results suggest that the differences between achene and receptacle samples may reveal significant distinctions in the transcriptomes of these tissues across various stages of development (Supplemental Fig.\u0026nbsp;1.). To identify differentially expressed transcripts between achene and receptacle, we performed DESeq2 analysis using a significance cut-off padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The larger number of DEGs was identified in the comparison between achene and receptacle at the MG stage (32,686 DEGs total; 17,608 up-regulated and 15,078 down-regulated). In contrast, the comparison between achene and receptacle at the R stage yielded the smallest number of DEGs, totaling 7,131 DEGs from which 6,217 were up-regulated and 914 were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Supplemental Fig.\u0026nbsp;2 and Supplemental Table\u0026nbsp;3). Venn diagrams were generated for pairwise comparisons between achene and receptacle at each stage (SG, MG, LG, W, TR, and R), and a total of 2,892 genes were found to be expressed in all six developmental stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Moreover, stage-specific analyses revealed that 3,612, 4,396, 3,069, 1,583, 817, and 189 genes were uniquely expressed at SG, MG, LG, W, TR and R stages, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Intriguingly, our findings reveal a notable decrease in the number of differentially expressed genes, particularly during the later developmental stages, including the W, TR, and R stages. Our results also showed a significant increase in the expression levels of achene-specific transcripts across the different fruit developmental stages. Additionally, the expression patterns of achene or receptacle-specific genes varied throughout different developmental stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These observations highlight the substantial expression of achene-specific transcripts during fruit maturation. The observed variation in differentially gene expression patterns between achenes and receptacles during fruit maturation can be attributed to their distinct tissue compositions. Achenes, as more complex organs consisting of the embryo, endosperm, and seed coat, naturally display greater complexity and a higher number of differentially expressed genes. Conversely, the receptacle, a simpler organ composed of cortex and pith, shows less variability in gene expression due to its more uniform tissue structure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eDynamic differential gene expression in achene and receptacle during fruit development\u003c/h2\u003e \u003cp\u003eTo elucidate the functional dynamics of gene expression between achene and receptacle tissues across six developmental stages, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using the ShinyGO web tool. We used a total of 2,892 DEGs identified across all different developmental stages of achene and receptacle (padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and subjected them to GO analysis to determine their associated Biological Process (BP), Cell Components (CC), and Molecular Functions (MF) associated with each gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplemental Table\u0026nbsp;4). Within the BP category, DEGs between achene and receptacle common to each of the six developmental stages were primarily involved in pathways including the phenylpropanoid metabolic process, post-embryonic development, phenylpropanoid biosynthesis, as well as lignin metabolism and biosynthesis. Of particular note, we observed a common expression pattern of genes related to the response to hormone (GO:0009725) across all six stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplemental Table\u0026nbsp;4). Additionally, in the CC category, the DEGs were found to relate to cellular components such as the vacuole, bounding membrane of organelle, vacuolar membrane, plant-type vacuole, and Golgi apparatus, among others (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Supplemental Table\u0026nbsp;4). Interestingly, in the MF category, a significant enrichment of transcription factors and DNA binding activities was observed, including DNA-binding transcription factor activity, transcription regulator activity, transcription cis-regulatory region binding, transcription regulatory region nucleic acid binding, and sequence-specific DNA binding (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Taken together, our findings suggest a conservation of transcriptional regulation mechanisms in the DEGs of achene and receptacle in octoploid strawberry. We also performed a KEGG pathway enrichment analysis on the 2,892 overlapped stage candidate DEGs identified. The primary signaling pathways associated with strawberry in the comparison between achene and receptacle include: Biosynthesis of secondary metabolites (139), Phenylpropanoid biosynthesis (31), Plant hormone signal transduction (32), Cutin, suberine and wax biosynthesis (9), stilbenoid, diarylheptanoid, and gingerol biosynthesis (4), Diterpenoid biosynthesis (6), Fatty acid degradation (9), and Flavonoid biosynthesis (6) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Supplemental Table\u0026nbsp;5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of genes involved in auxin homeostasis in achene and receptacle during fruit development of octoploid strawberry\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe expression levels of genes associated with auxin homeostasis were determined in achene and receptacle, and a total of 164 genes were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplemental Table\u0026nbsp;6). The TAAs and YUCs are essential components for the biosynthesis of the major natural auxin in plants, IAA. As the IPyA pathway mediated by TAAs and YUCs produces the majority of free IAA, the expression of TAAs and YUCs is indispensable for various essential developmental processes, including fruit development. In the octoploid strawberry \u0026lsquo;Royal Royce\u0026rsquo;, we identified \u003cem\u003eTAA1\u003c/em\u003e homoeologous gene copies located in the four subgenome of chromosome 4, \u003cem\u003eFxa4Ag102498, Fxa4Bg102453, Fxa4Cg202161\u003c/em\u003e, and \u003cem\u003eFxa4Dg101990\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among these copies, we found that \u003cem\u003eFxa4Bg102453, Fxa4Cg202161\u003c/em\u003e, and \u003cem\u003eFxa4Dg101990\u003c/em\u003e had high expression levels at the SG stage of achenes. Moreover, we identified homoeologous gene copies of \u003cem\u003eTAR1/2\u003c/em\u003e, including \u003cem\u003eFxa5Ag200524\u003c/em\u003e, \u003cem\u003eFxa5Bg100503\u003c/em\u003e, \u003cem\u003eFxa5Cg200481\u003c/em\u003e, and \u003cem\u003eFxa5Dg200494\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). All four copies showed high expression levels only during the SG and MG stages. For \u003cem\u003eTAR3/4\u003c/em\u003e, four homoeologous gene copies, \u003cem\u003eFxa2Ag101303\u003c/em\u003e, \u003cem\u003eFxa2Bg201139\u003c/em\u003e, \u003cem\u003eFxa2Cg202166\u003c/em\u003e, and \u003cem\u003eFxa2Dg201128\u003c/em\u003e were identified. Interestingly, only both \u003cem\u003eFxa2Cg202166\u003c/em\u003e and \u003cem\u003eFxa2Dg201128\u003c/em\u003e were highly expressed in the receptacle, particularly during the MG stage, compared to the achene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, four \u003cem\u003eFaYUC\u003c/em\u003e genes, \u003cem\u003eFaYUC2\u003c/em\u003e, \u003cem\u003eFaYUC4\u003c/em\u003e, \u003cem\u003eFaYUC10\u003c/em\u003e and \u003cem\u003eFaYUC11\u003c/em\u003e, were found in the octoploid strawberry. Protein analysis using the \u003cem\u003eFvYUC2\u003c/em\u003e sequence from diploid strawberry showed high similarity with four homoeologous copies of \u003cem\u003eFaYUC2\u003c/em\u003e, \u003cem\u003eFxa1Ag100425, Fxa1Bg200419, Fxa1Cg100379\u003c/em\u003e, and \u003cem\u003eFxa1Dg200387\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The gene expression result showed that only \u003cem\u003eFxa1Cg100379\u003c/em\u003e appears to be dominantly expressed at the LG stage at achene, while the expression of the other three copies is either absent or very low. For \u003cem\u003eFaYUC4\u003c/em\u003e, interestingly, all four homoeologous copies of \u003cem\u003eFxa2Ag102968, Fxa2Bg202798, Fxa2Cg201097\u003c/em\u003e, and \u003cem\u003eFxa2Dg202579\u003c/em\u003e located in chromosome 2 are highly expressed during the MG stage of achene development. However, the expression of \u003cem\u003eFaYUC4\u003c/em\u003e was only found at the subgenome A (\u003cem\u003eFxa2Ag102968\u003c/em\u003e), and B (\u003cem\u003eFxa2Bg202798\u003c/em\u003e) at the SG and LG stages. The gene \u003cem\u003eFaYUC10\u003c/em\u003e has four homoeologous copies, \u003cem\u003eFxa2Ag102487\u003c/em\u003e, \u003cem\u003eFxa2Bg202327\u003c/em\u003e, \u003cem\u003eFxa2Cg203233\u003c/em\u003e and \u003cem\u003eFxa2Dg202176\u003c/em\u003e, in all subgenomes of chromosome 2. The expression levels were constitutively higher during the SG, MG, and LG stages of the achene compared to other stages. In receptacle, \u003cem\u003eFxa2Ag102487\u003c/em\u003e, \u003cem\u003eFxa2Bg202327\u003c/em\u003e, and \u003cem\u003eFxa2Cg203233\u003c/em\u003e show relatively higher expression levels at the SG and MG stages. For \u003cem\u003eFaYUC11\u003c/em\u003e, three homoeologous gene copies, \u003cem\u003eFxa4Ag101793\u003c/em\u003e, \u003cem\u003eFxa4Bg101727\u003c/em\u003e, and \u003cem\u003eFxa4Cg201512\u003c/em\u003e, were found and showed higher expression in all developmental stages of the achene compared to the receptacle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, we identified high expressions of \u003cem\u003eGH3\u003c/em\u003e genes, which belong to one of the major auxin-responsive gene families. \u003cem\u003eFaGH3.1\u003c/em\u003e, \u003cem\u003eFaGH3.5\u003c/em\u003e, \u003cem\u003eFaGH3.6a\u003c/em\u003e, \u003cem\u003eFaGH3.6b\u003c/em\u003e, \u003cem\u003eFaGH3.9\u003c/em\u003e, and \u003cem\u003eFaGH3.17\u003c/em\u003e showed expression levels with LogFC values from 0.48 to 6.06 (average\u0026thinsp;=\u0026thinsp;2.4) at the SG stage and from 0.57 to 7.97 (average\u0026thinsp;=\u0026thinsp;2.67) at the MG stage in achenes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplemental Tables\u0026nbsp;3 and 6). It has been known that \u003cem\u003eGH3s\u003c/em\u003e play crucial roles in auxin homeostasis through conjugating free auxin with amino acids [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. We identified that the \u003cem\u003eFaGH3.5\u003c/em\u003e homoeologous gene copies, \u003cem\u003eFxa6Ag104259\u003c/em\u003e, \u003cem\u003eFxa6Bg103912\u003c/em\u003e, \u003cem\u003eFxa6Cg103796\u003c/em\u003e, and \u003cem\u003eFxa6Dg103714\u003c/em\u003e, were highly expressed in the achene during the SG and MG stages. Interestingly, only one copy of \u003cem\u003eFaGH3.6\u003c/em\u003e (\u003cem\u003eFxa3Ag100357\u003c/em\u003e) was highly expressed in receptacle during the SG and MG stages compared to the achene. Furthermore, \u003cem\u003eFaGH3.6a\u003c/em\u003e, \u003cem\u003eFaGH3.6b\u003c/em\u003e, \u003cem\u003eFaGH3.9\u003c/em\u003e, and \u003cem\u003eFaGH3.17\u003c/em\u003e showed predominantly high expression levels at the SG and MG stages of achene. Particularly, the three homoeologous copies of \u003cem\u003eFaGH3.6b\u003c/em\u003e, \u003cem\u003eFxa3Bg201817\u003c/em\u003e, \u003cem\u003eFxa3Cg101795\u003c/em\u003e, and \u003cem\u003eFxa3Dg201745\u003c/em\u003e, showed high expression levels in the achene at the SG stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor the PIN family genes, six homologs of PIN 1, 2, 4, 5, 8, and 10 were identified in \u003cem\u003eF. \u0026times; ananassa\u003c/em\u003e utilizing \u003cem\u003eF. vesca\u003c/em\u003e as a query. Among them, \u003cem\u003eFaPIN1\u003c/em\u003e, \u003cem\u003eFaPIN4\u003c/em\u003e, \u003cem\u003eFaPIN5\u003c/em\u003e, and \u003cem\u003eFaPIN8\u003c/em\u003e exhibited stronger expression in the receptacle at SG than in achene at SG, but \u003cem\u003eFaPIN10\u003c/em\u003e showed the opposite pattern. Homologs of AUX/LAX genes, including \u003cem\u003eAUX/LAX2\u003c/em\u003e, \u003cem\u003eAUX/LAX3\u003c/em\u003e, and \u003cem\u003eAUX/LAX4\u003c/em\u003e, were identified in \u003cem\u003eF. \u0026times; ananassa\u003c/em\u003e. While \u003cem\u003eFaAUX/LAX2\u003c/em\u003e exhibited high gene expression at MG of achene. \u003cem\u003eFaAUX/LAX4\u003c/em\u003e showed stronger gene expression at SG and MG of achene compared to receptacle (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We also discovered nine \u003cem\u003eAux/IAA\u003c/em\u003e genes and 14 ARF gene family homologs in \u003cem\u003eF. \u0026times; ananassa\u003c/em\u003e. Among them, \u003cem\u003eFaAux/IAA26b\u003c/em\u003e displayed differences in gene expression between achene and receptacle at MG. \u003cem\u003eFaAux/IAA27a\u003c/em\u003e and \u003cem\u003eFaAux/IAA27b\u003c/em\u003e showed slight differences in expression between achene and receptacle at SG and MG. Notably, at SG and MG, \u003cem\u003eFaARF3\u003c/em\u003e, \u003cem\u003eFaARF6a\u003c/em\u003e, \u003cem\u003eFaARF19a\u003c/em\u003e, and \u003cem\u003eFaARF19b\u003c/em\u003e gene families exhibited higher expression in receptacle than in achene. However, \u003cem\u003eFaARF1b\u003c/em\u003e displayed higher expression in achene from MG to W, and some genes including \u003cem\u003eFaARF16a\u003c/em\u003e, \u003cem\u003eFaARF16b\u003c/em\u003e, and \u003cem\u003eFaARF17a,c\u003c/em\u003e showed higher expression in SG of achene compared to receptacle. Lastly, TIR1, AFB2, and AFB5 homologs were also identified in \u003cem\u003eF. \u0026times; ananassa\u003c/em\u003e. These auxin biosynthesis-related genes influence various aspects of \u003cem\u003eF. \u0026times; ananassa\u003c/em\u003e fruit effects, including growth, perception of auxin, and more, throughout the stages from early to late, suggesting their multifaceted impact on the fruit development of octoploid strawberry (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTranscription factors involved in auxin biosynthesis signaling networks between achenes and receptacles during fruit development\u003c/h2\u003e \u003cp\u003eTo further understand auxin biosynthesis and transport between achene and receptacle, we used the entire list of DEG and clustered them across all six developmental stages based on their transcriptional expression. We identified four distinctive patterns of expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). Cluster 1 was formed by 1209 genes with increasing expression in achene having the highest expression at LG. Genes in cluster 1 have a low or no expression in the six receptacle developmental stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Cluster 2 formed by 1229 genes showed a decreased expression from achene to receptacle in a developmental manner. The more advanced the developmental stage the lower the expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Cluster 3 showed an opposite pattern as cluster 1, having lower expression in the achene and higher expression in the receptacle (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Finally, cluster 4 formed by 177 genes, showed variable expression across developmental stages in achene and receptacles samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince cluster 1 showed opposite developmental expression between achene and receptacle, we selected the entire set of genes identified in this cluster to conduct a correlation analysis to pinpoint transcription factors with shared expression profiles with genes involved in auxin biosynthesis and transport between achene and receptacle. A large portion of the strawberry genes are described as uncharacterized or unknown. Therefore, we removed them from the analysis. After removal of unrooted genes, the remaining genes were clustered using an edge confidence of at least 0.7. Our analysis yielded six main hubs containing transcription factors associated with auxin related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These transcription factor hubs were grouped into NAC/WYKR (blue), MYB (green), APETALA2/Ethylene Responsive Factor (purple), homeobox (orange), heat shock transcription factor (red) and bZIP (black). Since we were interested in auxin biosynthesis and transport, we identified the auxin related genes (yellow) that clustered together with other transcription factors. Three out of the six hubs contained auxin related genes and were clustered with the NAC/WYKR, bZIP and APETALA2/Ethylene Responsive Factor hubs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). \u003cem\u003eFvYUC10\u003c/em\u003e (\u003cem\u003eFxa2Ag102487\u003c/em\u003e) and \u003cem\u003eFaPIN10\u003c/em\u003e (\u003cem\u003eFxa4Ag100605\u003c/em\u003e) clustered together with the APETALA2/Ethylene Responsive Factor group. \u003cem\u003eFvARF1a\u003c/em\u003e (\u003cem\u003eFxa5Dg202383\u003c/em\u003e) and \u003cem\u003eFvARF16a\u003c/em\u003e (\u003cem\u003eFxa6Dg101291\u003c/em\u003e) co-expressed with the NAC/WYKR hub and \u003cem\u003eFvARF11\u003c/em\u003e (\u003cem\u003eFxa5Dg200932\u003c/em\u003e) with the bZIP group of transcription factor. Taken together, our analysis identifies potential transcription factors involved in the regulation of auxin and transport between achene and receptacles.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe plant hormone auxin plays a fundamental role in regulating cell expansion and cell division [32, 33]. Previous studies indicate that fruit development is intricately linked to achene growth, with the maturation of fleshy fruits being inhibited in the absence of achene. This repression is relieved when fertilized achenes release auxin signals to the ovary wall and receptacle [34]. In strawberries, achenes are considered the principal sites for auxin accumulation, playing a pivotal role in regulating auxin distribution across the entire fruit, thereby influencing its development. [11, 21, 35]. Studies on auxin in wild diploid strawberry, \u003cem\u003eFragaria vesca\u003c/em\u003e, have shown that auxin increases both the width and length of the fruit during the early stages of fruit development, while gibberellic acid (GA), mainly promotes vertical growth [17].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuxin Dynamics in Achene-Derived Auxin on Receptacle Growth and Strawberry Fruit Development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;Perkins-Veazie classified the ripening stages of strawberry fruit into four stages (green, white, pink, and red), with pink being reported as the turning point of ripening [8]. However, given that several previous studies have indicated the significant role of auxin synthesis and transport in the early developmental stages of strawberry growth, our research has not only divided the developmental process into six distinct stages, from early development to maturity but also separated achene and receptacle at each stage for hormone measurement and transcriptome analysis (Fig. 1). A previous study has reported a consistent reduction in IAA as the fruit transitions from the small green stage to the red stage, as shown in mixed tissue of achene and receptacle [36]. In this study, when analyzing the auxin content separately in the achene and receptacle of octoploid strawberries, we observed a significantly higher concentration of auxin in the achene compared to the receptacle, as observed in Fig. 1. This suggests that receptacle enlargement is likely due to auxin moving from achenes. Our results corroborate those of Estrada-Johnson et al. (2017) and Gu et al. (2019), who observed elevated IAA levels in achenes compared to receptacles during fruit development. Our study, however, distinguishes itself by employing a high-quality reference genome to perform detailed tissue-specific analyses across six developmental stages in octoploid strawberries, whereas Estrada-Johnson et al. (2017) focused on four stages. Moreover, while Gu et al. (2019) examined the transcriptome and auxin-related genes in diploid strawberries, our analysis extends to octoploid strawberry varieties, providing a more comprehensive understanding of auxin dynamics across different genomic complexities [28, 37]. One intriguing finding is the presence of approximately 50 - 110 pmol/g FW of IAA in the receptacles at the small green stage. This supports the result of temporal variations in auxin distribution, where auxin is synthesized within the achene and transported to the receptacle. Although present at a lower concentration compared to the achene, the IAA in the receptacle likely contributes to the coordination of fruit development, potentially regulating processes such as cell division and expansion. This supports the notion that even with a lower concentration, the receptacle serves as a receiver and transporter of IAA to modulate the growth and development of the strawberry fruit.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExploring Auxin Metabolism, Transport, Signaling, and Response Pathways in Achene and Receptacle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the regulation of this phenomenon by specific genes, we conducted a genome-wide comprehensive analysis of the auxin-specific transcriptome in the achene and receptacle of octoploid strawberries using the recently developed high-quality haplotype-phased reference genome for octoploid strawberries [38]. We compared and analyzed the transcriptomes separately for the achene and receptacle at each developmental stage. According to previous research, strawberry fruit set is completed within two to four days after fertilization, and it has been shown that transcriptional regulation and signaling metabolic changes occur at fertilization. This pattern is consistent with our results (Fig. 1 and Fig. 4) [11]. To determine the collaborative action of gene families involved in the biosynthesis and transport of auxin, we conducted a DEG analysis using the criteria under padj \u0026lt; 0.05, log2(fold change) \u0026gt; 1 to identify significantly differentially expressed genes at each developmental stage. In this study, we examined the expressions of homologous genes previously studied, such as TAA, TAR, YUC, GH3, Aux/IAA, TIR/AFB, and ARF, and found that the genes related to auxin biosynthesis were highly expressed during SG or MG in the achene. Throughout the comprehensive analysis in this study, it was found that genes such as TAA1, YUC4, and GH3.6b play a pivotal role in auxin biosynthesis primarily within the achene. Additionally, the PIN gene family, essential for auxin efflux facilitation, markedly impacts auxin\u0026apos;s directional flow owing to their unique intracellular localization [39, 40]. In octoploid strawberries, particularly during the SG stage, the expression of specific PIN genes (\u003cem\u003eFaPIN4\u003c/em\u003e, \u003cem\u003eFaPIN5\u003c/em\u003e, and \u003cem\u003eFaPIN8\u003c/em\u003e) is substantially elevated in the receptacle compared to the achene. This reaffirms the coordination of achene and receptacle in fruit TAA development. Previous studies have also reported the high expression of auxin-conjugating GH3 genes in ghost and ovary walls at the \u003cem\u003eF. vesca\u003c/em\u003e [11]. Furthermore, the presence of IAA amide conjugates and highly abundant IAA-protein conjugates have been reported in the receptacle of strawberries (Archbold and Dennis 1984; Park, et al. 2006). One notable aspect to consider is that in many species, a multitude of Aux/IAA proteins modulate ARF-mediated transcription and offer extensive signaling interactions in various processes involving auxin [41]. In this research, we discovered that gene copies within the Aux/IAA family, specifically \u003cem\u003eFaAux/IAA26a\u003c/em\u003e (\u003cem\u003eFxa2Ag103448\u003c/em\u003e, \u003cem\u003eFxa2Bg203233\u003c/em\u003e, \u003cem\u003eFxa2Cg200618\u003c/em\u003e, \u003cem\u003eFxa2Dg203024\u003c/em\u003e), \u003cem\u003eFaAux/IAA27a\u003c/em\u003e (Fxa1Ag100497, \u003cem\u003eFxa1Bg200482\u003c/em\u003e, Fxa1Cg100463, \u003cem\u003eFxa1Dg200444\u003c/em\u003e), and \u003cem\u003eFaAux/IAA27b\u003c/em\u003e (\u003cem\u003eFxa6Ag103081\u003c/em\u003e, \u003cem\u003eFxa6Bg102848\u003c/em\u003e, \u003cem\u003eFxa6Cg102729\u003c/em\u003e, \u003cem\u003eFxa6Dg102648\u003c/em\u003e), alongside TIR/AFB genes such as \u003cem\u003eFaTIR1\u003c/em\u003e (\u003cem\u003eFxa2Ag102496\u003c/em\u003e, \u003cem\u003eFxa2Bg202336\u003c/em\u003e, \u003cem\u003eFxa2Cg203243\u003c/em\u003e, \u003cem\u003eFxa2Dg202184\u003c/em\u003e), \u003cem\u003eFaAFB2\u003c/em\u003e (\u003cem\u003eFxa6Ag105135\u003c/em\u003e), and \u003cem\u003eFaAFB5\u003c/em\u003e (\u003cem\u003eFxa5Ag203451\u003c/em\u003e, \u003cem\u003eFxa5Bg103235\u003c/em\u003e, \u003cem\u003eFxa5Cg202953\u003c/em\u003e, \u003cem\u003eFxa5Dg203023\u003c/em\u003e), and ARF genes including \u003cem\u003eFaARF3\u003c/em\u003e (\u003cem\u003eFxa3Dg20069\u003c/em\u003e, \u003cem\u003eFxa3Cg100717\u003c/em\u003e, \u003cem\u003eFxa3Ag100785\u003c/em\u003e), \u003cem\u003eFaARF6a\u003c/em\u003e (\u003cem\u003eFxa3Cg100416\u003c/em\u003e, \u003cem\u003eFxa3Ag100462\u003c/em\u003e), \u003cem\u003eFaARF19a\u003c/em\u003e (\u003cem\u003eFxa1Cg100787\u003c/em\u003e, \u003cem\u003eFxa1Dg200710\u003c/em\u003e), and \u003cem\u003eFaARF19b\u003c/em\u003e (\u003cem\u003eFxa4Ag100540\u003c/em\u003e, \u003cem\u003eFxa4Bg100521\u003c/em\u003e, \u003cem\u003eFxa4Cg200482\u003c/em\u003e, \u003cem\u003eFxa4Dg100445\u003c/em\u003e), demonstrated notably higher expression levels in the receptacle than in the achene. This differential expression was particularly evident during the early developmental stages, from SG to LG, respectively. This intricate network of interactions may stem from gene expression driven by specific TIR/AFB, Aux/IAA, and ARF transcription factors in response to auxin [42].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterizing Auxin Transcription factors in Achene and Receptacle\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur study specifically aime aimed to elucidate the transcription factors associated with the genes discovered during fruit development in octoploid strawberry. We focused on transcription factors that exhibit co-expression patterns with genes involved in auxin biosynthesis and transport within both achene and receptacle tissues. Our analysis revealed that several auxin-related genes were grouped alongside the NAC/WYKR, bZIP, and AP2/ERF transcription factor hubs. Specifically, \u003cem\u003eFaYUC10\u003c/em\u003e (Fxa2Ag102487) and \u003cem\u003eFaPIN10\u003c/em\u003e (Fxa4Ag100605) were associated with the AP2/ERF cluster. Auxin has been proposed to indirectly promote fruit ripening by stimulating the transcription of several ethylene components, leading to ethylene-induced fruit ripening and softening. Additionally, \u003cem\u003eFaARF1a\u003c/em\u003e (Fxa5Dg202383) and \u003cem\u003eFaARF16a\u003c/em\u003e (Fxa6Dg101291) demonstrated co-expression with the NAC/WYKR hub, while \u003cem\u003eFaARF11\u003c/em\u003e (Fxa5Dg200932) aligned with the bZIP transcription factor group. This collective data suggests potential transcription factors that may play a crucial role in regulating auxin biosynthesis and its transport dynamics between achene and receptacle during strawberry fruit ripening.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn our study provides comprehensive gene expression profiling specific at subgenome level of octoploid strawberries from early to late fruit developmental stages. This information highlights an important yet insufficiently explored area in the field of octoploid strawberry auxin research. Furthermore, identifying pattens of subgenome-specific gene expression would implicate pathways of auxin metabolites, as well as the transport and perception of auxin between achenes and receptacles (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Leveraging recently available high-quality haplotype-phased reference genomes and genome-wide transcriptome profiling analysis, we were able to unveil the network of genes in auxin homeostasis and enhance our understanding of the regulatory mechanisms during fruit development in strawberry.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSample preparations\u003c/h2\u003e \u003cp\u003eThe samples of the strawberry fruits (\u003cem\u003eFragaria\u003c/em\u003e \u0026times; \u003cem\u003eananassa\u003c/em\u003e Duch. cv. Brilliance) were used for transcriptome analysis. The six different developmental stages of harvested fruits corresponded that stage 1 is Small Green (SG), stage 2 is Medium Green (MG), stage 3 is Large Green (LG), stage 4 is White (W), stage 5 is Turning Red (TR), and stage 6 is Red (R). In early January, the fruits were harvested from the UF strawberry field at Gulf Coast Research and Education Center in Balm, Florida. All stages of the fruits, including transcriptome sequencing biological replicated, were harvested simultaneously. Using the forceps and scalpels, each stage of the achenes and receptacle were separated from the fruits.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAuxin measurement\u003c/h2\u003e \u003cp\u003eApproximately 20 mg (fresh weight) of achene and receptacle samples from 6 fruit stages were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C. For IAA extraction and purification, frozen samples of achenes and receptacles were ground with pestles in liquid nitrogen and promptly submerged in 1.1 mL sodium phosphate buffer (50mM, pH 7.0) containing 0.1% diethyl dithiocarbamic acid sodium salt and 10 ng/mL of [\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e]-IAA as internal standard. Samples were incubated at 4\u0026deg;C with continuous shaking for 40 min and then centrifuged at 13000 \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 15 min. After collecting supernatant and adjusting pH to 2.7, samples were purified by solid-phase extraction using Oasis\u0026trade; HLB columns (WAT094225; Waters, MA, USA). The final elutes with 80% methanol were evaporated to dryness \u003cem\u003ein vacuo\u003c/em\u003e and stored at -20\u0026deg;C until LC/MS analysis.\u003c/p\u003e \u003cp\u003eThe IAA detection method using liquid chromatography and mass spectrometer (LC-MS) was adapted from [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. All samples were resuspended in MilliQ water and analyzed using Vanquish Horizon ultra-high performance liquid chromatography (UHPLC) installed with an Eclipse Plus C18 column (2.1 \u0026times; 50 mm, 1.8 \u0026micro;m) (Agilent) and mass analysis was performed using a TSQ Altis Triple Quadrupole (Thermo Scientific) MS/MS system with an ion funnel. MRM parameters of the standards (precursor m/z, fragment m/z, radio frequency (RF) lens and collision energy) of each compound were optimized on the machine using direct infusion of the authentic standards. IAA and [\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e]-IAA were purchased from Cambridge Isotope Laboratories. For IAA detection, the mass spectrometer was operated in positive ionization mode at an ion spray voltage of 4800 V. Formic acid (0.1%) in water and 100% acetonitrile were used as mobile phases A and B, respectively, with a gradient program (0\u0026ndash;95% solvent B over 4 min) at a flow rate of 0.4 mL per min. The sheath gas, aux gas and sweep gas were set at 50, 9 and 1 (arbitrary units), respectively. Ion transfer tube temperature and vaporizer temperature were set at 325\u0026deg;C and 350\u0026deg;C, respectively. For MRM monitoring, both Q1 and Q3 resolutions were set at 0.7 FWHM with collision-induced dissociation (CID) gas at 1.5 mTorr. The scan cycle time was 0.8 s. MRM for IAA was used to monitor parent ion \u0026rarr; product ion reactions for each analyte as follows: m/z 175.983\u0026rarr;130.071 (CE, 18 V) for IAA; m/z 182.091\u0026rarr;136 (CE, 18 V) for [\u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e]-IAA. IAA analysis was conducted with three biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptome data analysis\u003c/h2\u003e \u003cp\u003eThe total RNA from separate achene and receptacle at six development stage with three replications per sample were extracted by the Spectrum\u0026trade; Plant Total RNA Kit (Sigma-Aldrich, MO, USA) as fallowing the manufacturer protocol. To generate RNA-seq for illumina sequencing library were fallowed Illumina sequencing protocol. The resulting sequencing library were performed pair-end sequenced (2 x 150bp) by Illumina NovaSeq instruments at Novogene Bioinformatics Institute, Beijing, China. Raw read sequences obtained from 36 sequenced libraries were quality trimmed and filtered using Trimmomatic [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Data quality was assessed using FastQC. Trimmed paired end reads were aligned to the \u0026lsquo;Royal Royce\u0026rsquo; octoploid genome [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] using Hisat2 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Differentially expressed genes (DEG) analysis was conducted using the DESeq2 package in R script [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] using a fold change \u0026gt;|1| and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (after the false discovery rate adjustment for multiple testing (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for the null hypothesis. Total number of reads mapped to each gene was used to calculate transcripts per million (TPM) values, which were determined using a custom Python script.\u003c/p\u003e \u003cp\u003eGene Ontology (GO) enrichment analysis was conducted using Arabidopsis gene information provided by the Royal Royce genome annotation database [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The analysis was conducted through the ShinyGO tool (version 0.77, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/go/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/go/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]), applying a P-value cutoff of \u0026le;\u0026thinsp;0.05 (FDR) and default options.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eConfirmation of homology of octoploid strawberry auxin hormone pathway genes\u003c/h2\u003e \u003cp\u003eGenes associated with auxin biosynthesis were explored in the octoploid strawberry involved utilizing known genes from \u003cem\u003eF. vesca\u003c/em\u003e and previous literature. BlastP was performed with significant criteria under e-value\u0026thinsp;=\u0026thinsp;0, pident\u0026thinsp;\u0026lt;\u0026thinsp;90, bit score\u0026thinsp;\u0026gt;\u0026thinsp;100 based on the protein sequence against the Royal Royce reference genome [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] for each hormone-related gene [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eK-means Clustering and Gene Co-expression Analysis\u003c/h2\u003e \u003cp\u003eTranscripts per million (TPM) counts were used as input for the K-means clustering analysis. TPM values were averaged for the replicates of each tissue (achene or receptacle) at any given stage (Stage 1 to Stage 6). Averaged TPM values were normalized to log\u003csub\u003e2\u003c/sub\u003e(TPM\u0026thinsp;+\u0026thinsp;1), scaled, and used as input for clustering analysis. DEGs were categorized into four clusters using the k-means algorithm implemented using the R programming language. In order to identify transcriptional correlations among genes with shared expression profile analysis achene or receptacle, the \u003cem\u003ecor\u003c/em\u003e function was implemented in the R package WGCNA. Then, genes with shared expression profiles were considered as seed candidates and were used to build a gene co-expression analysis to obtain direct and indirect interactions. A high confidence score of 0.7 was used as a threshold [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Only high levels of confidence interactions were considered as valid as used in the final gene co-expression analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAbscisic Acid (ABA)\u003c/p\u003e\n\u003cp\u003eApetala2/Ethylene Responsive Factor (AP2/ERF)\u003c/p\u003e\n\u003cp\u003eAuxin Response Factor (ARFs)\u003c/p\u003e\n\u003cp\u003eAuxin/Indole-3-Acetic Acid Proteins (Aux/IAAs)\u003c/p\u003e\n\u003cp\u003eBasic Region/ Leucine Zipper Motif (bZIP)\u003c/p\u003e\n\u003cp\u003eBiological Process (BP)\u003c/p\u003e\n\u003cp\u003eCell Components (CC)\u003c/p\u003e\n\u003cp\u003eGene Ontology (GO)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGretchen Hagen 3 (GH3s)\u003c/p\u003e\n\u003cp\u003eIndole-3-Acetic Acid (IAA)\u003c/p\u003e\n\u003cp\u003eIndole-3-Pyruvic Acid (IPyA)\u003c/p\u003e\n\u003cp\u003eKyoto Encyclopedia Of Genes And Genomes (KEGG)\u003c/p\u003e\n\u003cp\u003eLarge Green (LG)\u003c/p\u003e\n\u003cp\u003eMedium Green (MG)\u003c/p\u003e\n\u003cp\u003eMolecular Functions (MF)\u003c/p\u003e\n\u003cp\u003eNAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY),\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhenylacetic Acid (PAA)\u003c/p\u003e\n\u003cp\u003ePin-Formed (PINs)\u003c/p\u003e\n\u003cp\u003ePrincipal Component Analysis (PCA)\u003c/p\u003e\n\u003cp\u003eRed (R)\u003c/p\u003e\n\u003cp\u003eRoyal Royce (FaRR1)\u003c/p\u003e\n\u003cp\u003eSmall Green (SG)\u003c/p\u003e\n\u003cp\u003eTaa-Related (TAR)\u003c/p\u003e\n\u003cp\u003eTransport Inhibitor Response 1 (TIR1)\u003c/p\u003e\n\u003cp\u003eTransport Inhibitor Response 1 / Auxin-Signaling F-Box (TIR/AFBs)\u003c/p\u003e\n\u003cp\u003eTryptophan Aminotransferase Of Arabidopsis (TAA)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTurning Red (TR)\u003c/p\u003e\n\u003cp\u003eWhite (W)\u003c/p\u003e\n\u003cp\u003eYUCCA (YUCs)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-throughput sequencing data analyzed in present study are available under NCBI BioProject\u0026nbsp;PRJNA1010111.\u0026nbsp;The online version contains Supporting Information available\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research is supported by grants from the United States Department of Agriculture National Institute of Food and Agriculture (NIFA) Specialty Crops Research Initiative (SCRI) \u0026ldquo;Delivering Breeding and Management Solutions to Prevent Losses to Emerging and Expanding Disease Threats in Strawberry\u0026rdquo; under award number (#2022-51181-38328) to S.L and \u003cem\u003eNSF-IOS-CAREER-\u003c/em\u003e 2142898 to J.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eYJ,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eKB, JK and SL contributed to the study design and drafted the article. YJ, JK, TK, KB, HH, ML, ZL, VW, SL and analyzed the experiment results, prepared figures and tables. All authors read and approved the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe extend our gratitude to Dr. Vance M. Whitaker for providing the fruit materials used in this study. The authors thank for the technical support and assistance with fruit preparations provided by Dr. Youngjae Oh and Sadikshya Sharma. Also, we thank Ru Dai and Veronica Perez for their technical support for auxin quantification.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHardigan MA, Feldmann MJ, Lorant A, Bird KA, Famula R, Acharya C, Cole G, Edger PP, Knapp SJ: \u003cstrong\u003eGenome synteny has been conserved among the octoploid progenitors of cultivated strawberry over millions of years of evolution\u003c/strong\u003e. \u003cem\u003eFront Plant Sci. \u003c/em\u003e2020, \u003cstrong\u003e10\u003c/strong\u003e:1789.\u003c/li\u003e\n\u003cli\u003eEdger PP, Poorten TJ, VanBuren R, Hardigan MA, Colle M, McKain MR, Smith RD, Teresi SJ, Nelson AD, Wai CM: \u003cstrong\u003eOrigin and evolution of the octoploid strawberry genome\u003c/strong\u003e. \u003cem\u003eNat. 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applications\u003c/strong\u003e. \u003cem\u003eBMC Bioinformatics \u003c/em\u003e2008, \u003cstrong\u003e10\u003c/strong\u003e:421.\u003c/li\u003e\n\u003cli\u003eBegcy K, Nosenko T, Zhou L-Z, Fragner L, Weckwerth W, Dresselhaus T: \u003cstrong\u003eMale sterility in maize after transient heat stress during the tetrad stage of pollen development\u003c/strong\u003e. \u003cem\u003ePlant Physiol. \u003c/em\u003e2019, \u003cstrong\u003e181\u003c/strong\u003e(2):683-700.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Auxin, Strawberry fruit, Plant hormone, Achene, Receptacle, Transcriptomics","lastPublishedDoi":"10.21203/rs.3.rs-4589609/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4589609/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe plant hormone auxin plays a crucial role in regulating important functions in strawberry fruit development. Although a few studies have described the complex auxin biosynthetic and signaling pathway in wild diploid strawberry (\u003cem\u003eFragaria vesca\u003c/em\u003e), the molecular mechanisms underlying auxin biosynthesis and crosstalk in octoploid strawberry fruit development are not fully characterized. To address this knowledge gap, comprehensive transcriptomic analyses were conducted at different stages of fruit development and compared between the achene and receptacle to identify developmentally regulated auxin biosynthetic genes and transcription factors during the fruit ripening process. Similar to wild diploid strawberry, octoploid strawberry accumulates high levels of auxin in achene compared to receptacle.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eGenes involved in auxin biosynthesis and conjugation, such as Tryptophan Aminotransferase of Arabidopsis (TAAs), YUCCA (YUCs), and Gretchen Hagen 3 (GH3s), were found to be primarily expressed in the achene, with low expression in the receptacle. Interestingly, several genes involved in auxin transport and signaling like Pin-Formed (PINs), Auxin/Indole-3-Acetic Acid Proteins (Aux/IAAs), Transport Inhibitor Response 1 / Auxin-Signaling F-Box (TIR/AFBs) and Auxin Response Factor (ARFs) were more abundantly expressed in the receptacle. Moreover, by examining DEGs and their transcriptional profiles across all six developmental stages, we identified key auxin-related genes co-clustered with transcription factors from the NAM-ATAF1,2-CUC2/ WRKYGQK motif (NAC/WYKY), Basic Region/ Leucine Zipper motif (bZIP), and APETALA2/Ethylene Responsive Factor (AP2/ERF) groups.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese results elucidate the complex regulatory network of auxin biosynthesis and its intricate crosstalk within the achene and receptacle, enriching our understanding of fruit development in octoploid strawberries.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Gene Network Uncover Temporal and Spatial Changes of Genes in Auxin Homeostasis During Fruit Development in Strawberry (F. ×ananassa)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-05 02:15:15","doi":"10.21203/rs.3.rs-4589609/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-08T06:27:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-05T15:42:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-01T09:07:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124972319318533007423462764644946229554","date":"2024-06-21T00:28:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155649062850048741239943776860967770561","date":"2024-06-20T01:24:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-19T13:37:34+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-19T11:02:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-19T10:58:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-19T10:57:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-06-16T12:01:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a0e37ca9-f6f0-4b38-abb9-1774b1a64701","owner":[],"postedDate":"July 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-23T16:01:31+00:00","versionOfRecord":{"articleIdentity":"rs-4589609","link":"https://doi.org/10.1186/s12870-024-05577-5","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-09-20 15:57:23","publishedOnDateReadable":"September 20th, 2024"},"versionCreatedAt":"2024-07-05 02:15:15","video":"","vorDoi":"10.1186/s12870-024-05577-5","vorDoiUrl":"https://doi.org/10.1186/s12870-024-05577-5","workflowStages":[]},"version":"v1","identity":"rs-4589609","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4589609","identity":"rs-4589609","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}