Interactions of JAZ Repressors with Anthocyanin Biosynthesis-Related Transcription Factors of Fragaria × ananassa

: Strawberry fruits are rich in ﬂavonoids like proanthocyanidins and anthocyanins. Their biosynthesis and accumulation are controlled by the MYB-bHLH-WD40 (MBW) transcriptional complex, which is mainly formed by basic helix-loop-helix (bHLH) and MYB transcription factors (TFs). In Arabidopsis thaliana both bHLH and MYB TFs are repressed by JASMONATE ZIM-DOMAIN (JAZ) proteins, the key repressors of the jasmonate-signaling pathway. The aim of this research was the characterization of the FaJAZ1 / 8.1 / 9 / 10 proteins and molecular targets of signaling components and anthocyanin biosynthesis-related TFs of Fragaria × ananassa by protein–protein interactions. For this, domain compositions were studied by multiple alignments and phylogenetic analyses, while interactions were analyzed by yeast two-hybrid (Y2H) assays. We detected high conservation of FaJAZ proteins and jasmonate-signaling components, as well as FabHLHs and FaMYB10 TFs. Moreover, we report the F. × ananassa YABBY1 (FaYAB1) TF, which is related to anthocyanin biosynthesis in Arabidopsis, showed high conservation of functional domains. We demonstrated that FaJAZ repressors interacted with F. × ananassa NOVEL INTERACTOR OF JAZ (FaNINJA), FaMYC2, and JASMONATE ASSOCIATED MYC2-LIKE (FaJAM) proteins. Besides, transcription factors of MBW-complex like FabHLH3, FabHLH33, and FaMYB10, together with FaYAB1, were molecular targets of FaJAZ repressors, exhibiting speciﬁcity or redundancy of interaction depending on particular FaJAZ protein. Overall, these results suggest that interactions of jasmonate-signaling components are fully conserved, and anthocyanin biosynthesis might be regulated by JAZ repressors in F. × ananassa . according to expected for plants of Rosaceae family [60]. These results indicate the structural conservation of YAB1 proteins in F . × ananassa .


Introduction
Fleshy fruits of cultivated strawberry (Fragaria × ananassa Duch.) are highly valued by its taste, flavor, red color, and their multiple nutritional properties [1]. The nutritional value is given by flavonoid compounds like anthocyanins, which are in large amounts in red fruits [2] and have different benefits for human health [1]. In turn, other flavonoids like proanthocyanidins (PAs) are in a higher quantity at the immature stage of strawberry fruits [2], participating in the protection against pathogens of flowers and early fruit tissues [3] and also with antioxidant benefits for humans [4]. and the Jas domain allowing the interaction with a high number of MYC and JAM TFs through JAZ-interaction domain (JID) domain [31,41]. Moreover, protein-protein interactions of JAZ to regulate some processes such as the secondary metabolism are also mediated by the Jas domain, which interacts with the bHLH domain of TT8 and EGL3, the CT domain of PAP1/MYB75 in Arabidopsis [22,42]. In some cases, TIFY is the main domain involved in the interaction with the C domain of NINJA protein [33,41] or with YABBY1 (YAB1) [20], although the TF domain involved in this interaction has not yet been reported. Finally, in M. × domestica the repressor role of JAZs-bHLH3 regulating the PA and anthocyanin biosynthesis have been demonstrated [10].
According to previously mentioned, this research aimed to characterize the interaction of strawberry JAZ proteins (FaJAZ1, FaJAZ8.1, FaJAZ9, and FaJAZ10) with canonical JA-signaling components (FaNINJA,FaMYC2s,and FaJAMs), as well as MBW complex-related components (bHLHs and MYBs). Specially, we characterize for the first time a YABBY gene in strawberry (FaYAB1) at the sequence level and the protein-protein interactions with FaJAZs. We found functional conservation of FaJAZ proteins with JA signaling-and anthocyanin biosynthesis-related TFs and proteins.

Identification of Ortholog Sequences
Full-length proteins of A. thaliana were obtained from TAIR database (v10, https://www.arabidopsis. org/, last accessed date: 16 June 2020) and were used as queries to search the ortholog sequences not previously characterized for F. vesca, M. × domestica, Vitis vinifera L., and Solanum lycopersicum L. in RefSeq Protein database (https://www.ncbi.nlm.nih.gov/refseq/, last accessed date: 16 June 2020) by BLASTP search using default parameters. Output sequences with higher identity, query cover, and lower e-value were selected as orthologs in each species. The Genebank Accession numbers are listed in Table S1.

Isolation and Cloning of Encoding Sequences
Total RNA was extracted from 4 g of strawberry fruits using the cetyltrimethylammonium bromide (CTAB) method and further RNA purification by RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to previously reported [14,39]. The cDNA synthesis was performed using the RevertAid H Minus First Strand cDNA Synthesis Kit (ThermoScientific, Waltham, MA, USA) from 1 µg of total RNA, according to the manufacturer's instructions. Full-length coding sequences (CDS) of JA-signaling (NINJA, MYC2, MYC2-like, JAM1, JAM2, JAZ9) and YAB1 of F. vesca, and anthocyanin-related transcription factors (bHLH3, bHLH33, MYB10, MYB1, MYB5) of F. × ananassa were used for primer design containing attB1 and attB2 sites at 5' and 3' ends (Table S2), respectively, required for cloning by Gateway technology (Invitrogen, Carlsbad, CA, USA). Isolation of coding sequence was performed from cDNA by reverse transcription-PCR (RT-PCR) using Phusion Hot Start II high-fidelity DNA Polymerase (ThermoScientific, Waltham, MA, USA). The PCR conditions were as follows: 98 • C for 30 s; 35 cycles of 98 • C for 10s, 58 • C for 30 s and 72 • C for 30 s per kb; and 72 • C for 10 min. All PCR products were analyzed by 1% (w/v) agarose gel and purified using Zymoclean Gel DNA Recovery Kit' (Zymo Research, Irvine, CA, USA).

Results and Discussion
Fleshy fruits of strawberry are rich in anthocyanins, which gives high nutritional value and antioxidant capacity to human health [1,4]. Although many aspects of the metabolism and biochemical pathways of the anthocyanin biosynthesis are well known during the development of strawberry fruits, the knowledge about the role of some molecular pathways regulating its biosynthesis is still scarce. Some phytohormones are responsible for their biosynthesis and accumulation, such as ABA [13]. However, recently, the existence of possible regulatory mechanisms of the JA-signaling pathway through the interaction of JAZ repressors with TFs related to anthocyanin biosynthesis and accumulation in M. × domestica [10] and A. thaliana [19,21] have been demonstrated. In strawberry, anthocyanin accumulation is promoted by exogenous MeJA and a putative role of its JA-signaling pathway [14,15] have been proposed. In this work, we report that the JA-signaling pathway, through ortholog sequences for JAZ1, JAZ8.1, JAZ9, and JAZ10 repressors in F. × ananassa ( Figure S1a), targets anthocyanin biosynthesis-related TFs suggesting that JAZs might be another regulator for anthocyanin accumulation in strawberry fruit.

The JA-Signaling Pathway is Structurally and Functionally Conserved in Fragaria × ananassa
The activation of JA-signal transduction triggers the regulation of developmental processes and responses to multiple stresses [23,39,40]. Essentially, the JA-signaling pathway regulates other molecular pathways by protein-protein interactions mediated by JAZ repressors and MYC TFs, and it is well described in Arabidopsis [29][30][31][32] and starting to be known in strawberry [15,27,28,39]. On the one hand, F. vesca and F. × ananassa contain a conserved perception mechanism at the structural and functional Agronomy 2020, 10, 1586 5 of 15 levels [27,28]. However, on the other hand, components for downstream JA-signaling pathway like MYC or JAM TFs have only been characterized at the transcriptional level in F. × ananassa [39].
Noteworthy, most JAZ proteins are transcriptional repressors and are characterized by the presence of TIFY and Jas domains [29,35,36,41]. Although, some JAZ sequences like JAZ7 and JAZ8 contain a variant degron sequence avoiding the recognition by COI1 coreceptor and the continuous repression of MYC TFs for a fine-tuning of JA responses in Arabidopsis [47]. In F. vesca 12 JAZ proteins have been identified [27,37,39], and JAZ1, JAZ8.1, and JAZ10 orthologs were also recently characterized in F. × ananassa, showing high conservation in their structural and functional domains [28]. In this work, we isolated and characterized JAZ9 of F. × ananassa, which showed highly conserved TIFY and Jas domains ( Figure S2a,b), similarly to F. vesca and A. thaliana orthologs [39], indicating that JAZ proteins maintain functional domains and possibly repression functions by protein-protein interactions with TFs in F. × ananassa.
Regarding other JA-signaling components, we detected high conservation for interaction domains of NINJA, MYC2, MYC2-like, JAM1, and JAM2 proteins in F. × ananassa ( Figures S3-S5), similar to that reported for F. vesca [39], A. thaliana [33], and Gossypium hirsutum [48] among others. On one side, the NINJA adaptor is a corepressor of the JA-signaling pathway [33]. We isolated the F. × ananassa NINJA protein, which is the A. thaliana orthologous sequence ( Figure S1b) and exhibited high conservation of amino acid residues in EAR and C domains ( Figure S3a,b) for corepressor interaction of JA-signaling pathway with TOPLESS (TPL) and JAZ proteins, respectively [33]. According to this, F. × ananassa NINJA interacted with FaJAZ1, FaJAZ9, and FaJAZ10 ( Figure 1), similar to that reported in Arabidopsis [33]. In contrast, FaJAZ8.1 did not display interaction with FaNINJA ( Figure 1) according to previous reports in Arabidopsis orthologs through Y2H assays [33]. On the other side, MYC TFs displayed the highest conservation level for the bHLH domain ( Figure S4c), which is involved in the binding to G-box regions of the DNA [31,49], and it is according with their roles as master regulators in the promotion of JA responses and anthocyanin accumulation in Arabidopsis leaves [31]. In F. vesca, MYC2 and MYC2-like were reported by Garrido-Bigotes et al. [39] and correspond to FvbHLH80 and FvbHLH96 [50], respectively. In the case of the JID domain, which is involved in the interaction with Jas domain of JAZ repressors [31] a lower amino acid residue conservation was detected because this domain is lacking in V. vinifera MYC2-like ortholog sequence ( Figure S4a). An evident interaction was detected between all FaJAZs and FaMYC2/FaMYC2-like (Figure 1), similar to that reported for MYC2 ortholog in Arabidopsis [31]. Finally, JAM1/bHLH017, JAM2/bHLH013, and JAM3/bHLH003 are negative regulators of JA-associated responses in Arabidopsis and have been reported as inhibitors for anthocyanin accumulation [34,51]. In strawberry, these proteins contained JID and bHLH domains, like MYC TFs [31], but lack the conserved transactivation domain (TAD) (Figures S5 and S6) compared to MYC TFs [31]. Protein-protein interactions were observed between FaJAM1, FaJAM2, and all FaJAZ proteins (Figure 1), similar to previously reported in A. thaliana. It is important to highlight that the cotransformation with the controls constituted by pGADT7-(AD)-TFs and pGBKT7-(DBD) empty vector did not exhibit the activation of ADE2 or HIS3 reporter genes ( Figure S7). Globally, the structural conservation of key domains in FaNINJA, FaMYC2, FaMYC2-like, FaJAM1, and FaJAM2 ( Figures S3-S5) as well as in FaJAZ1, FaJAZ8.1, FaJAZ9, and FaJAZ10 ( Figure S2), together with the interaction observed by Y2H assays indicate that the repressor capacity of JAZ proteins would be conserved in F. × ananassa.

bHLH and MYB Transcription Factors Interact with of JAZ Repressors in Fragaria × ananassa
The biosynthesis of anthocyanins is controlled by the MBW transcriptional complex in higher plants, including A. thaliana, M. × domestica, and F. × ananassa [5][6][7][8][9][10]42] between others. This complex is constituted by R2R3-MYB, bHLH, and WD40 TFs [5], and in F. × ananassa have been characterized two major complexes formed by different TFs and regulating differentially the PAs and anthocyanin biosynthesis [6] being MYB10 component, the key TF for anthocyanin accumulation in strawberry [7]. Besides, recent findings suggest that some TFs associated with PA biosynthesis like bHLH3 may be involved in the anthocyanin biosynthesis [8,15]. Besides, the two MYB1 and MYB5 TFs act as repressors for the positive action of the MBW complex in the anthocyanin accumulation [6]. Noteworthy, F. × ananassa bHLH3, bHLH33, and MYB10 TFs have their orthologs TT8, EGL3, and PAP1/MYB75, respectively [5], which are molecular targets of JAZ proteins in A. thaliana [22].
In the present research, we isolated bHLH3, bHLH33, and MYB10 in F. × ananassa which showed high conservation of bHLH and CT domains ( Figure S8a-c), and identity close to 100% (Table S4) according to previously characterized TFs in cultivated strawberry [6]. An important difference In the present research, we isolated bHLH3, bHLH33, and MYB10 in F. × ananassa which showed high conservation of bHLH and CT domains ( Figure S8a-c), and identity close to 100% (Table S4) according to previously characterized TFs in cultivated strawberry [6]. An important difference between our results compared to Schaart et al. [6], was the isolation of a larger sequence of FabHLH33 ( Figure S9), although the key bHLH domain is completely conserved ( Figure S8b). Early studies suggest that bHLH3/MYB10 and bHLH33/MYB10 regulate the transcriptional activation of LAR/ANR and ANS/UFGT genes in F. × ananassa [6,8]. However, previous studies suggest that FaMYB10 in association with FabHLH3 could regulate the activation of ANS/UFGT transcription for anthocyanin biosynthesis [15]. In the present research, FabHLH3 showed interaction with FaJAZ8.1, FaJAZ9, and FaJAZ10 (Figure 2), and this TF is regulated by the same JAZ repressors and JAZ1 orthologs in Arabidopsis [22]. Otherwise, the interaction of JAZ2, JAZ3, and JAZ8 proteins with bHLH3 ortholog in M. × domestica was also reported [10]. Surprisingly, FabHLH33 only showed interaction with FaJAZ1 ( Figure 2), while A. thaliana ortholog shows a clear interaction with JAZ8.1, JAZ9, and JAZ10 [22]. Possibly, the additional N-ter sequence presented in the FabHLH33 sequence isolated in this research ( Figure S8) could interfere in the interaction with FaJAZ8.1, FaJAZ9, and FaJAZ10. In the case of FaMYB10, the interaction with FaJAZ1, FaJAZ8.1, and FaJAZ9 were observed (Figure 2), similar to that reported for its ortholog proteins in A. thaliana [22]. Finally, FaMYB1 and FaMYB5 (Figures S10 and S11) are R2R3 MYB TFs involved in the decrease of anthocyanin accumulation in strawberry fruits [52,53]. These negative TFs did not exhibit interaction with any JAZ protein in F. × ananassa (Figure 2), in contrast to that observed for some A. thaliana R2R3 related TFs like MYB21 and MYB24 that interact with JAZ1, JAZ8, and JAZ11 [54]. However, it is noteworthy, that Arabidopsis orthologs MYB6 ( Figure S10e) and MYB5 ( Figure S11d) are phylogenetically distant from MYB21, MYB24, and MYB10 transcription factors [55]. Moreover, the absence of interaction of FaMYB1 and FaMYB5 with JAZ repressors (Figure 2) may be because these proteins lack the CT interaction domain (Figures S10 and S11), which is also observed for MYB10, MYB21, and MYB24 [54]. Overall, JAZ proteins show specific and redundant roles [40] for interaction with MBW complex regulation, although other protein-protein interactions mediated by the other FaJAZ repressors described previously [39] should be evaluated.
( Figure S9), although the key bHLH domain is completely conserved ( Figure S8b). Early studies suggest that bHLH3/MYB10 and bHLH33/MYB10 regulate the transcriptional activation of LAR/ANR and ANS/UFGT genes in F. × ananassa [6,8]. However, previous studies suggest that FaMYB10 in association with FabHLH3 could regulate the activation of ANS/UFGT transcription for anthocyanin biosynthesis [15]. In the present research, FabHLH3 showed interaction with FaJAZ8.1, FaJAZ9, and FaJAZ10 (Figure 2), and this TF is regulated by the same JAZ repressors and JAZ1 orthologs in Arabidopsis [22]. Otherwise, the interaction of JAZ2, JAZ3, and JAZ8 proteins with bHLH3 ortholog in M. × domestica was also reported [10]. Surprisingly, FabHLH33 only showed interaction with FaJAZ1 ( Figure 2), while A. thaliana ortholog shows a clear interaction with JAZ8.1, JAZ9, and JAZ10 [22]. Possibly, the additional N-ter sequence presented in the FabHLH33 sequence isolated in this research ( Figure S8) could interfere in the interaction with FaJAZ8.1, FaJAZ9, and FaJAZ10. In the case of FaMYB10, the interaction with FaJAZ1, FaJAZ8.1, and FaJAZ9 were observed (Figure 2), similar to that reported for its ortholog proteins in A. thaliana [22]. Finally, FaMYB1 and FaMYB5 (Figures S10 and S11) are R2R3 MYB TFs involved in the decrease of anthocyanin accumulation in strawberry fruits [52,53]. These negative TFs did not exhibit interaction with any JAZ protein in F. × ananassa (Figure 2), in contrast to that observed for some A. thaliana R2R3 related TFs like MYB21 and MYB24 that interact with JAZ1, JAZ8, and JAZ11 [54]. However, it is noteworthy, that Arabidopsis orthologs MYB6 ( Figure S10e) and MYB5 ( Figure S11d) are phylogenetically distant from MYB21, MYB24, and MYB10 transcription factors [55]. Moreover, the absence of interaction of FaMYB1 and FaMYB5 with JAZ repressors (Figure 2) may be because these proteins lack the CT interaction domain ( Figures S10 and S11), which is also observed for MYB10, MYB21, and MYB24 [54]. Overall, JAZ proteins show specific and redundant roles [40] for interaction with MBW complex regulation, although other protein-protein interactions mediated by the other FaJAZ repressors described previously [39] should be evaluated.

The Transcription Factor YAB1 Interacts with JAZ Repressors in Fragaria × ananassa
Some TF-associated gene families have specific roles in development processes or stresses, but the major TFs show additional functions. For instance, the YABBY gene family is constituted by six TFs regulating the development of leaves and flowers [56,57]. However, some studies reported that the YAB1/FIL gene is related to anthocyanin biosynthesis through the control of the upregulation of the PAP1/MYB75 gene, while JAs through JAZ proteins are regulators of YAB1 in Arabidopsis [20]. Thus, YAB1 is an additional regulator of anthocyanin accumulation profile in Arabidopsis [20], and its presence and conservation in strawberry should be of great value for understanding more in-depth the regulation of anthocyanin biosynthesis.
As the YABBY gene family in strawberry is still unknown, we characterized and isolated YAB1 (Figure 3), which is the ortholog of YAB1/FIL in A. thaliana ( Figure S1c) [20], YAB3 in V. vinifera [58], YAB1a and YAB1b in S. lycopersicum [59]. This protein family contains C2C2-zinc finger and YABBY domains [57], which displayed high conservation in F. × ananassa (Figure 3a,b), as well as orthologs of other fruits species like M. × domestica, V. vinifera, and S. lycopersicum. In detail, the conservation of amino acid residues is near to 100% with their ortholog sequences (Table S4), in both C2C2 ( Figure 3a) and YABBY (Figure 3b) domains, similar to reported for its orthologs in other fruits like V. vinifera [58] and S. lycopersicum [59]. Moreover, FaYAB1 was clustered near to F. vesca YAB1s and M. × domestica YAB1a-like (Figure 3c), according to expected for plants of Rosaceae family [60]. These results indicate the structural conservation of YAB1 proteins in F. × ananassa.

The Transcription Factor YAB1 Interacts with JAZ Repressors in Fragaria × ananassa
Some TF-associated gene families have specific roles in development processes or stresses, but the major TFs show additional functions. For instance, the YABBY gene family is constituted by six TFs regulating the development of leaves and flowers [56,57]. However, some studies reported that the YAB1/FIL gene is related to anthocyanin biosynthesis through the control of the upregulation of the PAP1/MYB75 gene, while JAs through JAZ proteins are regulators of YAB1 in Arabidopsis [20]. Thus, YAB1 is an additional regulator of anthocyanin accumulation profile in Arabidopsis [20], and its presence and conservation in strawberry should be of great value for understanding more indepth the regulation of anthocyanin biosynthesis.
As the YABBY gene family in strawberry is still unknown, we characterized and isolated YAB1 (Figure 3), which is the ortholog of YAB1/FIL in A. thaliana ( Figure S1c) [20], YAB3 in V. vinifera [58], YAB1a and YAB1b in S. lycopersicum [59]. This protein family contains C2C2-zinc finger and YABBY domains [57], which displayed high conservation in F. × ananassa (Figure 3a,b), as well as orthologs of other fruits species like M. × domestica, V. vinifera, and S. lycopersicum. In detail, the conservation of amino acid residues is near to 100% with their ortholog sequences (Table S4), in both C2C2 ( Figure  3a) and YABBY (Figure 3b) domains, similar to reported for its orthologs in other fruits like V. vinifera [58] and S. lycopersicum [59]. Moreover, FaYAB1 was clustered near to F. vesca YAB1s and M. × domestica YAB1a-like (Figure 3c), according to expected for plants of Rosaceae family [60]. These results indicate the structural conservation of YAB1 proteins in F. × ananassa.  Table S1.
The interaction YABs-JAZs was reported in Arabidopsis between JAZ1, JAZ3, JAZ4, JAZ9 repressors, and YAB1, however, only JAZ3 shows a strong interaction [20]. We detected a weak interaction of FaJAZ1-FaYAB1 ( Figure 4) similar to previously reported [20], but a stronger interaction was observed with FaJAZ9 ( Figure 4). It is important to highlight that the AtJAZ3 ortholog is not present in F. × ananassa, however, FaJAZ9 is the closest ortholog [39], therefore might be supplying the Agronomy 2020, 10, 1586 9 of 15 repressor activity of AtJAZ3 in F. × ananassa. Globally, these interactions suggest that F. × ananassa YAB1 ortholog could be regulated by JAZ9 protein.
Agronomy 2020, 10,x FOR PEER REVIEW 9 of 15 interaction of FaJAZ1-FaYAB1 ( Figure 4) similar to previously reported [20], but a stronger interaction was observed with FaJAZ9 ( Figure 4). It is important to highlight that the AtJAZ3 ortholog is not present in F. × ananassa, however, FaJAZ9 is the closest ortholog [39], therefore might be supplying the repressor activity of AtJAZ3 in F. × ananassa. Globally, these interactions suggest that F. × ananassa YAB1 ortholog could be regulated by JAZ9 protein.

Conclusions
In summary, interactions of JAZ proteins with components of the JA-signaling pathway and TFs related to anthocyanin biosynthesis were presented. F. × ananassa JAZ repressors are conserved at the structural level, including JAZ1, JAZ8.1, JAZ9, and JAZ10. Additionally, these proteins interact with TFs like MYC2, MYC2-like, JAM1, and JAM2, together with the NINJA adaptor protein in the same manner to that observed for A. thaliana orthologs, indicating that the function of the JA-signaling pathway would be conserved in F. × ananassa. JAZ proteins target TFs involved in the anthocyanin biosynthesis and showed functional redundancy, indicating anthocyanin biosynthesis could be finetuning by the JA-signaling pathway. Otherwise, MYB1 and MYB5 do not interact with JAZ1, JAZ8.1, JAZ9, or JAZ10, showing that JAZ repressors would not regulate these negative regulators of the anthocyanin accumulation. Finally, YAB1 protein was characterized for the first time in strawberry, which is a molecular target of FaJAZ9, suggesting that the anthocyanin biosynthesis could also be regulated by interactions between JAZs and YAB1 in F. × ananassa. Furthermore, these results provide additional information about JAZ-molecular targets related to anthocyanin biosynthesis in strawberry fruits. However, further studies will be necessary to know the putative in vivo regulation of the anthocyanin accumulation by JAZ repressors during the ripening of strawberry fruit.

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1; Table S1

Conclusions
In summary, interactions of JAZ proteins with components of the JA-signaling pathway and TFs related to anthocyanin biosynthesis were presented. F. × ananassa JAZ repressors are conserved at the structural level, including JAZ1, JAZ8.1, JAZ9, and JAZ10. Additionally, these proteins interact with TFs like MYC2, MYC2-like, JAM1, and JAM2, together with the NINJA adaptor protein in the same manner to that observed for A. thaliana orthologs, indicating that the function of the JA-signaling pathway would be conserved in F. × ananassa. JAZ proteins target TFs involved in the anthocyanin biosynthesis and showed functional redundancy, indicating anthocyanin biosynthesis could be fine-tuning by the JA-signaling pathway. Otherwise, MYB1 and MYB5 do not interact with JAZ1, JAZ8.1, JAZ9, or JAZ10, showing that JAZ repressors would not regulate these negative regulators of the anthocyanin accumulation. Finally, YAB1 protein was characterized for the first time in strawberry, which is a molecular target of FaJAZ9, suggesting that the anthocyanin biosynthesis could also be regulated by interactions between JAZs and YAB1 in F. × ananassa. Furthermore, these results provide additional information about JAZ-molecular targets related to anthocyanin biosynthesis in strawberry fruits. However, further studies will be necessary to know the putative in vivo regulation of the anthocyanin accumulation by JAZ repressors during the ripening of strawberry fruit.