Investigation of the JASMONATE ZIM-DOMAIN Gene Family Reveals the Canonical JA-Signaling Pathway in Pineapple

Simple Summary JASMONATE ZIM-DOMAIN (JAZ) proteins are key components of the jasmonate (JA) signaling pathway in response to biotic and abiotic stresses in plants. Information about the JAZ gene family in pineapple (AcJAZ) is limited. In this study, 14 AcJAZ genes were identified in the pineapple genome. A complete overview of AcJAZ genes is presented, including the chromosome locations, phylogenetic relationships, gene structures, conserved motifs and cis-regulatory elements, and expression patterns at different developmental stages and under various stress conditions, and their possible involvement in diverse functions is suggested. Furthermore, the BiFC analysis revealed direct binary interactions between AcJAZs and crucial JA-signaling regulators in vivo. These results suggest that AcJAZs and other vital players function in the JA-signaling pathway in response to abiotic stresses in pineapple. Abstract JASMONATE ZIM-DOMAIN (JAZ) proteins are negative regulators of the jasmonate (JA)-signaling pathway and play pivotal roles in plant resistance to biotic and abiotic stresses. Genome-wide identification of JAZ genes has been performed in many plant species. However, systematic information about pineapple (Ananas comosus L. Merr.) JAZ genes (AcJAZs) is still not available. In this study, we identified 14 AcJAZ genes and classified them into five groups along with the Arabidopsis and rice orthologs. The AcJAZ genes have 3–10 exons, and the putative AcJAZ proteins have between two and eight conserved regions, including the TIFY motif and Jas domain. The cis-acting element analysis revealed that the putative promoter regions of AcJAZs contain between three and eight abiotic stress-responsive cis-acting elements. The gene-expression analysis suggested that AcJAZs were expressed differentially during plant development and subjected to regulation by the cold, heat, salt, and osmotic stresses as well as by phytohormones. Moreover, the BiFC analysis of protein interactions among the central JA-signaling regulators showed that AcJAZ4, AcMYC2, AcNINJA, and AcJAM1 could interact with AcJAZ5 and AcJAZ13 in vivo, indicating a canonical JA-signaling pathway in pineapple. These results increase our understanding of the functions of AcJAZs and the responses of the core players in the JA-signaling pathway to abiotic stresses.

Moreover, pineapple plants have excellent stress tolerance to adverse conditions, including drought and salt stress. Since JA signaling plays a pivotal role in plant response to stresses and JAZ proteins are a core component of JA signaling, it is important to explore the JA-signaling pathway based on the JAZ gene family in pineapple (AcJAZ). In this study, the genes involved in the JA-signaling pathway were identified in pineapple, and an extensive investigation of the AcJAZs was conducted. These results provide a comprehensive understanding of the JA-signaling pathway in pineapple, which should facilitate further research related to pineapple stress response and tolerance.

Phylogenetic Analysis
The protein sequences of 14 AcJAZs, 13 AtJAZs, and 15 OsJAZs were aligned by MEGA7 using the Clustalw method. Then, the alignment file was used to construct a Neighbor-Joining-based phylogenetic tree with the bootstrap value of 1000. The phylogenetic tree was annotated using the online tool Evolview (https://evolgenius.info/ /evolview-v2/#login (accessed on 20 December 2021)). All protein sequences used for phylogenetic analysis are listed in Supplementary File S1.

Analysis of Conserved Motif and Gene Structure
To show sequence similarity between JAZ proteins of pineapple, 14 AcJAZ sequences were used to create a phylogenetic tree by MEGA7 and present conserved motifs and gene structures. The MEME software (Multiple EM for Motif Elicitation, http://meme-suite. org/tools/meme (accessed on 20 December 2021)) was used to identify conserved motifs with the maximum searching number set to 15. All motif information of pineapple JAZ proteins is listed in Supplementary File S2. Moreover, the exon-intron organization data were extracted from the Pineapple Genomics database (PGD). The visualization of the phylogenetic tree, conserved motifs, and gene structures was conducted with Tbtools [44].

BiFC Assay
The YFP-N end-tagged pSPYNE and YFP-C end-tagged pSPYCE vectors were used to transform Agrobacterium tumefaciens strain GV3101 and then infiltrate tobacco (Nicotiana benthamiana) leaves for BiFC (Bimolecular fluorescence complementation) assay. The tobacco infiltration and inflorescence observations were conducted following the procedure reported by Yuan and Xu [48]. Infusion strategies were adopted for vector preparation using CloneExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The primers used in this assay are listed in Table S1.

Fourteen JAZs Were Identified in the Pineapple Genome
The genome of pineapple variety F153 was used as a reference in this study [50]. In total, 14 JAZ proteins were identified based on BLASTP and HMM searching, and the corresponding genes were designated as AcJAZ1-AcJAZ14 based on their location on pineapple chromosomes from top to bottom, in which AcJAZ1-13 were located on ten pineapple chromosomes (chromosomes 01, 02, 05, 06, 07, 09, 12,14,19,20), and AcJAZ14 was located on an unanchored scaffold_1707 ( Figure 1A). Based on the DNA and amino acid sequences, the characteristics of pineapple JAZ genes and the putative proteins, including gene ID, chromosome locations, complete coding sequence (CDS) length, exon numbers, protein length, molecular weight, and isoelectric point (pI) are shown in Table 1. The AcJAZ genes possessed a minimum of three exons, and AcJAZ1 had the maximum (10) exons. The CDSs of AcJAZs ranged from 486 to 1608 bp in length, with the corresponding protein length ranging from 161 to 535 aa, the molecular weight from 16,867.08 Da to 56,897.76 Da, and the isoelectric point between 5.32 and 9.8 ( Table 1).

Protein Sequence Analysis Showed the Phylogenetic Relationship of AcJAZs
The evolutionary relationship among the pineapple JAZ proteins was studied by constructing a phylogenetic tree along with 13 Arabidopsis and 15 rice JAZ protein sequences using MEGA X, following the neighbor-joining (NJ) method ( Figure 1B). The JAZ proteins were divided into six groups according to the topology of the phylogenetic tree, and the AcJAZs were represented in five groups. In the first group, one OsJAZ (OsJAZ2), three AtJAZs (AtJAZ7, AtJAZ8, and AtJAZ13), and four AcJAZs (AcJAZ1, AcJAZ7, AcJAZ9, and AcJAZ14) were grouped. OsJAZ3, OsJAZ4, AtJAZ3, AtJAZ4, AtJAZ9, and AcJAZ4 belonged to the second group. The third group contained only three genes, i.e., OsJAZ5, AtJAZ10, and AcJAZ5. OsJAZ1, AtJAZ11-AtJAZ12, and six AcJAZs (AcJAZ2, AcJAZ6, AcJAZ8, AcJAZ10, AcJAZ11, and AcJAZ12) belonged to the fourth group. The fifth group was composed of AcJAZs (AcJAZ3 and AcJAZ13), AtJAZs (AtJAZ1, AtJAZ2, AtJAZ5, and AtJAZ6), and OsJAZ8. The sixth group only had OsJAZs (OsJAZ6, OsJAZ7, OsJAZ9-OsJAZ15) ( Figure 1B). The phylogenetic relationship of AcJAZs along with AtJAZs and OsJAZs indicated that those JAZ proteins shared a similarity of protein sequences and a common ancestor.

AcJAZs Are Conserved in Gene Structure and Motif Organization
To understand the gene structure and protein motif in a phylogenetic view, the JAZ proteins of pineapple were used to construct a phylogenetic tree, gene structure, and motif analysis in the same order as in the phylogenetic tree. In the absence of Arabidopsis and rice, the phylogenetic tree showed a similar relationship with the result described above (Figure 2A). According to the pineapple genome release (V3.0), the introns in AcJAZs ranged from two to nine, and exons ranged from 3 to 10. AcJAZ2 had the most extended genomic DNA sequence (12,295 bp) with the longest intron (fourth intron, 9049 bp), while AcJAZ4 had the shortest nucleotide sequence with the shortest intron (third intron, 53 bp). Most of the AcJAZ genes have the 3 UTR and 5 UTR annotated, with the exceptions of AcJAZ10 without 5 UTR, AcJAZ1 and AcJAZ2 without 3 UTR, and AcJAZ12 lacking both UTRs. AcJAZ7, AcJAZ9, and AcJAZ14 had similar structure and sequence lengths with two introns and three exons. AcJAZ7, AcJAZ8, AcJAZ9, AcJAZ10, AcJAZ11, AcJAZ12, and AcJAZ14 are rather high, indicating the strong nucleus localization of JAZ proteins of pineapple. Protein motifs might be assigned putative functions ofproteins. The motifs of pineapple JAZ proteins were identified using Multiple-Expectation maximization for Motif Elicitation (MEME) software. All AcJAZs harbored between two and eight motifs, while AcJAZ1, 2, 4, and 5 had two motifs (Figure 2A). Motif 1 and motif 2 were present in all AcJAZs, whereas motif 3 and motif 4 were present in five AcJAZs (Figure 2A). Two protein pairs, AcJAZ9/AcJAZ14 and AcJAZ3/AcJAZ13, had the same motif features (Figure 2A and Figure S2). Further, AcJAZ protein sequences were subjected to Pfam analysis to identify the conserved motifs, and we found nine Pfam motifs (tify, Jas_motif, CCT, F-box_4, FXR_C1, GATA, zf-Dof, Fz, and TFIIE-A_C) ( Figure S3). Motif 1 and motif 2 of the MEME analysis represented the tify and Jas domains, respectively ( Figure 2B), and motif 3 represented the GATA zinc finger domain, which uses four cysteine residues to coordinate a zinc ion. However, no Pfam information was detected for motifs 4 (PKKIRYTVRKEVALR) and 7 (VQAVLLLLGG), and they could be new motifs with unknown functions.
To investigate whether those JAZ proteins are localized in the nucleus, we analyzed the possible subcellular localization of AcJAZs and OsJAZ proteins using DeepLoc (https: //services.healthtech.dtu.dk/service.php?DeepLoc-1.0 (accessed on 20 December 2021)) and MULocDeep (http://mu-loc.org/ (accessed on 20 December 2021)). Both methods were consistent with each other in predicting that all 14 AcJAZs were localized in the nucleus (Table S2). As a comparison, we also analyzed 15 rice JAZ proteins. The DeepLoc analysis predicted that all 15 OsJAZs were localized in the nucleus, while MULocDeep predicted that 12 OsJAZs were localized in the nucleus and three OsJAZ were mainly localized elsewhere (Table S3). The prediction score of the nucleus localization of AcJAZ1 and AcJAZ3 is relatively low, while the those of the nucleus localization of AcJAZ2, AcJAZ7, AcJAZ8, AcJAZ9, AcJAZ10, AcJAZ11, AcJAZ12, and AcJAZ14 are rather high, indicating the strong nucleus localization of JAZ proteins of pineapple.

AcJAZs Are Differentially Expressed in Pineapple Organs
FPKM values calculated from RNA-seq generated from nine different tissues were used to create a heatmap and study the expression pattern of AcJAZ genes ( Figure 3A) [47]. In the samples analyzed, all the AcJAZ genes except AcJAZ12 were expressed. The clustering based on the expression patterns resulted in three groups. The first group consisted of five AcJAZs (AcJAZ1, AcJAZ2, AcJAZ4, AcJAZ6, and AcJAZ11) with relatively high expression levels. The second group had AcJAZ12, AcJAZ7, AcJAZ5, AcJAZ9, and AcJAZ14, with infrequent representation in the investigated tissues. The third group had four genes (AcJAZ3, AcJAZ8, AcJAZ10, and AcJAZ13) with moderate and varied expression levels. AcJAZ4 had the highest expression level among AcJAZ genes, and its expression level was higher in the calyx, petal, and fruit developmental stages 1-3, whereas AcJAZ12 was rarely expressed. AcJAZ7 showed in petal stage 3 (P3)-specific expression, and AcJAZ5 had a calyx (C1-C4)-specific expression. The expression patterns of AcJAZ8, AcJAZ9, AcJAZ10, and AcJAZ14 were constantly low in all tissues. Two genes, AcJAZ3 and AcJAZ13, had a similar expression pattern and were highly expressed in the calyx (C1-C3), petals (P2-P3), flowers, and leaves ( Figure 3A). The RNA-seq results were further verified for AcJAZ2, AcJAZ4, AcJAZ5, AcJAZ11, and AcJAZ13 expression using qPCR ( Figure 3B). Consistent with RNA-seq, the qPCR results also showed constitutively high expression of AcJAZ4 and low expression of AcJAZ5 in all the tissues. The expression patterns of these genes in petals, stamen, and fruit also showed a similar expression pattern as observed in the RNA-seq.   1-3), S1-S6 (Stamen stage 1-6), Flower, Fruit _S1-S7, leaf, root. The scale indicates log 10 transformed gene expression by colors ranging from red (high) to blue (low). (B) The expression of AcJAZ4, AcJAZ2, AcJAZ11, AcJAZ13, and AcJAZ5 in petal, stamen, and fruit determined by RT-qPCR. AcPP2A was used as the reference.

AcJAZs Are Involved in Responses to Phytohormone and Abiotic Stress
To predict the abiotic stress-responsive cis-elements, the PlantCARE online tool was used to analyze the upstream (2000 bp) sequences of 14 pineapple AcJAZ genes and nine putative abiotic stress-responsive cis-elements were detected ( Figure 4A). Each of the 14 AcJAZ genes had between three and eight putative responsive elements except for AcJAZ11, and all other AcJAZs had one or more ABREs ( Figure 4A, Table S4). Not surprisingly, all AcJAZs harbor at least one MeJA-responsive element in the 2kb upstream sequences, suggesting their functions in the JA-signaling pathway. The abscisic acid-responsive element (ABRE) and wound-responsive element (WRE3) were the first and third most abundant elements, present in 13 AcJAZ genes, suggesting the involvement of AcJAZ in the ABA and wounding response.  We further investigated the expression of AcJAZ genes in response to other hormonal and stress conditions. AcJAZ2, AcJAZ4, AcJAZ5, AcJAZ11, and AcJAZ13, representatives  (100 µM (a)), SA (100 µM (b)), IAA (100 µM (c)), ABA (100 µM (d)), 6-BA (100 µM (e)), NaCl (150 mM (f)), PEG4000 (15% (g)), Heat (45 • C (h)), and Cold (4 • C (i)). The leaves of the treated seedlings were used for the analysis. AcPP2A gene was used for RT-qPCR normalization. Data in (B) were analyzed using one-way ANOVA and post hoc Tukey test, and significant differences are indicated by different letters (upper case) at p < 0.01.
We further investigated the expression of AcJAZ genes in response to other hormonal and stress conditions. AcJAZ2, AcJAZ4, AcJAZ5, AcJAZ11, and AcJAZ13, representatives of different groups, were selected for this analysis. The results showed that their expression is regulated by different phytohormones (IAA, ABA, SA, and 6-BA) and abiotic stress treatments (cold, heat, salt, and osmotic stresses) ( Figure 4B). AcJAZ2 and AcJAZ5 were regulated significantly by SA, IAA, ABA, and 6-BA, and the expression of AcJAZ11 was induced considerably by 6-BA treatment. Under abiotic treatments, the expression of AcJAZ2 and AcJAZ5 was significantly induced by salt, osmotic, heat, and cold stresses. Furthermore, AcJAZ11 and AcJAZ13 were markedly induced by osmotic and cold treatments, respectively. Taken together, these results suggest that AcJAZs may play important roles in plant responses to phytohormone and abiotic stress, in addition to the JA signaling.

AcJAZs Respond to MeJA in Different Ways
JAZ proteins function as repressors of JA-responsive genes in JA signaling [51][52][53][54]. The cis-elements analysis showed that all of the JAZ genes have MeJA-responsive genes. Therefore, we investigated the expression levels of the 12 AcJAZs under MeJA treatment ( Figure 5). The results indicated that AcJAZ6, AcJAZ8, and AcJAZ12 were insensitive to MeJA treatment and their expression levels did not change compared to control. AcJAZ3, AcJAZ4, AcJAZ7, AcJAZ11, and AcJAZ13 were the early-responsive genes, with a high expression level after 2 h of treatment. AcJAZ2 and AcJAZ5 could be considered constant MeJA-responsive genes as their expressions were continuously increased at different time points after treatment. Interestingly, two genes AcJAZ9 and AcJAZ14, were repressed and showed a decreasing expression tendency after the treatment ( Figure 5).

Protein-Protein Interaction (PPI) Analysis Revealed the Conserved Interactions among the Central JA-Signaling Regulators in Pineapple
The transcription factor MYC2 plays a crucial role in the JA-signaling pathway, which drives the expression of JA-responsive genes [24,55,56], whereas JAZs bind with MYC2 and inhibit its activity, thereby turning off the expression of JA-responsive genes. We identified the pineapple MYC2 gene (Aco018875.1), AcMYC2 (Table S5), and investigated its interactions with AcJAZ4, AcJAZ5, AcJAZ11, and AcJAZ13 by BiFC. We found that only AcJAZ5 and AcJAZ13 interacted with AcMYC2 in nuclei of tobacco leaf cells ( Figure 6 and Figure S4, which confirmed the canonical function of JAZs in pineapple. In Arabidopsis, JAM1 is reported as a negative regulator of JA signaling that competitively binds to the promoters of JA-responsive genes and attenuates JA signaling [24,[57][58][59][60]. Furthermore, several JAZs could interact with JAM proteins and repress the expression of their targets such as those of MYC2 transcription factors. We verified this regulatory mechanism in pineapple by identifying the JAM1 (Aco005839.1) gene, AcJAM1 (Table S6), and validating its interactions with AcJAZs using BiFC. AcJAM1 and AcJAZs (AcJAZ5 and AcJAZ13) showed a clear interaction in BiFC experiments, and the signal was detected in nuclei of tobacco leaf cells ( Figure S4). Novel Interactor of JAZ (NINJA) is another protein that interacts with JAZs directly and recruits the co-repressor TOPLESS (TPL) [29]. Thus, the pineapple NINJA gene (Aco006735.1) AcNINJA was identified (Table S7), and the interactions between AcNINJA and AcJAZs were also investigated. The results showed that both AcJAZ5 and AcJAZ13 interacted with AcNINJA in nuclei of leaf cells, but no signal was detected from the interaction of AcNINJA with AcJAZ4 or AcJAZ11 (Figures 6 and S4 and Table S8). In addition, homo-and heteromeric interactions between JAZ proteins widely exist in different species, including Arabidopsis, rubber tree, and cotton [61][62][63]. We analyzed and detected the heteromeric interactions of AcJAZ4 with AcJAZ5 and AcJAZ13, but not AcJAZ11 ( Figure 6 and Table S8). The signal was localized in the cytoplasm and nuclei ( Figure 6). Taken together, the PPI analysis revealed the interactions among the central JA-signaling regulators in pineapple and suggested the conservation of the JA-signaling pathway in plants.

Discussion
The JA-signaling pathway is one of the critical pathways involved in plant response to abiotic and biotic stresses. The basic helix-loop-helix transcription factors MYC2, MYC3, MYC4, and MYC5, are key regulators in this pathway which initiate the expression of JA-responsive genes, whereas the JAM1 transcription factor negatively regulates the pathway [24,[55][56][57][58][59]. JAZ proteins repress MYC2 and JAM1 by interacting via the C-terminal Jas domain and N-terminal JIDS motif of MYC2 and JAM1. JAZ proteins also interact with NINJA and other JAZ proteins via the TIFY domain [61][62][63]. NINJA, a well-defined transcription co-repressor, interacts with TPL through its EAR domain to recruit the repressive CDK8 Mediator complex and Histone Deacetylase 19 (HD19), thus repressing the expression of target genes [59,[64][65][66]. Although the pineapple genome has been deciphered [50], the JA-signaling pathway in pineapple has not been well characterized. In this study, the main regulators involved in JA signaling, including 9 MYCs, 8 NINJAs, 5 JAM1s, and 14 JAZs, were identified in pineapple (Tables S5-S7). Based on the gene-expression analysis of the AcJAZ gene family (Figures 4 and 5) and protein-interaction results among the main regulators ( Figure 6), we propose a hypothetical model of the JA-signaling pathway in pineapple, which is similar to that of Arabidopsis ( Figure 7). However, it is worth mentioning that, AcJAZ2, AcJAZ4, AcJAZ5, AcJAZ11, and AcJAZ13 were the early JA-response genes in pineapple, and might play essential roles in pineapple JA signaling.
Biology 2022, 11, x FOR PEER REVIEW

Discussion
The JA-signaling pathway is one of the critical pathways involved in plant re to abiotic and biotic stresses. The basic helix-loop-helix transcription factors MYC3, MYC4, and MYC5, are key regulators in this pathway which initiate the expr of JA-responsive genes, whereas the JAM1 transcription factor negatively regula pathway [24,[55][56][57][58][59]. JAZ proteins repress MYC2 and JAM1 by interacting via the C nal Jas domain and N-terminal JIDS motif of MYC2 and JAM1. JAZ proteins also i with NINJA and other JAZ proteins via the TIFY domain [61][62][63]. NINJA, a well-d transcription co-repressor, interacts with TPL through its EAR domain to recruit pressive CDK8 Mediator complex and Histone Deacetylase 19 (HD19), thus repress expression of target genes [59,[64][65][66]. Although the pineapple genome has been phered [50], the JA-signaling pathway in pineapple has not been well characteriz this study, the main regulators involved in JA signaling, including 9 MYCs, 8 NIN JAM1s, and 14 JAZs, were identified in pineapple (Tables S5-S7). Based on the ge pression analysis of the AcJAZ gene family (Figures 4 and 5) and protein-interact sults among the main regulators ( Figure 6), we propose a hypothetical model of t signaling pathway in pineapple, which is similar to that of Arabidopsis (Figure 7) ever, it is worth mentioning that, AcJAZ2, AcJAZ4, AcJAZ5, AcJAZ11, and AcJAZ1 the early JA-response genes in pineapple, and might play essential roles in pineap signaling. Based on the F153 genome data [50], we identified 14 gene members in the pin JAZ gene family (Table 1). A total of 42 JAZ proteins from pineapple and two speci (15) and Arabidopsis (13), representing monocot and dicot plants, were used for co ative and phylogenetic analyses [67,68]. The number of JAZ proteins in the pineap nome was similar to those in rice and Arabidopsis, indicating that the JAZ gene fam Based on the F153 genome data [50], we identified 14 gene members in the pineapple JAZ gene family (Table 1). A total of 42 JAZ proteins from pineapple and two species, rice (15) and Arabidopsis (13), representing monocot and dicot plants, were used for comparative and phylogenetic analyses [67,68]. The number of JAZ proteins in the pineapple genome was similar to those in rice and Arabidopsis, indicating that the JAZ gene family in those three species might encounter similar genome-wide duplication events. According to the Neighbor-Joining (NJ) phylogenetic tree, the 42 JAZ proteins from three species were clustered into six groups ( Figure 1B). Group 6 only contained the JAZs from rice, while groups 1, 4, and 5 only had one JAZ from rice, reflecting the evolutionary differences among these three plants. There was a closer relationship between Arabidopsis and pineapple. Furthermore, the members in different pineapple JAZ gene family clusters showed significant divergences in exon-intron organization, sequence length, and motifs. Still, those in the same group were very similar for these characteristics (Figure 2A). In addition, the divergence in the motif composition and gene structure provided additional evidence to support phylogenetic groupings.
The JAZ proteins harbor two domains, Jas and TIFY, which interact either with MYC transcription factors when the JA-signaling pathway is not active or with the JA-Ile-SCF COI complex when the pathway is activated. However, the TIFY domain mediates the interactions with NINJA and the formation of homo-and hetero-dimers within the JAZ subfamily [61][62][63]. Pfam results revealed that these AcJAZ proteins possessed a conserved TIFY domain and a Jas domain ( Figure 2B). It has been reported that the TIFY domain has several deviations in plants [28,32]. Multiple sequence alignments indicated that the AcJAZ proteins also had several TIFY deviations ( Figure S1A). The core sequence of "TIFY" was replaced with "TISF" in AcJAZ6, "TLSF" in AcJAZ11 and AcJAZ12, "TLLF" in AcJAZ8, and "TLLY" in AcJAZ10 ( Figure S1A). The structural diversity of the core "TIFY" domain in pineapple JAZ proteins suggests functional diversity among the family members. In contrast, the sequences of the Jas domain among the JAZs in pineapple were more conserved ( Figure S1B). Most of the JAZ genes are also JA-responsive genes in the JA-signaling pathway. For instance, AtJAZ10 was expressed immediately after JA treatment [69,70]. The results from MeJA treatment indicate that AcJAZs are JA-responsive genes, among which AcJAZ3, AcJAZ4, AcJAZ7, AcJAZ11, and AcJAZ13 are transiently responsive, while AcJAZ2, AcJAZ5, and JAZ13 are constantly JA-responsive. It has been reported that OsJAZ8 could be induced by the JA signal to confer resistance to bacterial blight [71]. Phylogenetic analysis showed that AcJAZ3, AcJAZ13, and OsJAZ8 are in the same subcluster, suggesting that MeJA-induced AcJAZ3 and AcJAZ13 might also respond to biotic stress. Under MeJA treatment, the expression of AcJAZ3, AcJAZ4, AcJAZ7, AcJAZ11, and AcJAZ1 were transiently up-regulated, and that of AcJAZ2 and AcJAZ5 constantly up-regulated ( Figure 5). The JA-responsive expression of AcJAZ genes in pineapple also provides evidence for the negative feedback loop of the JA-signaling pathway.
The JA-signaling pathway has also been reported to be involved in various abiotic stresses. A previous study showed that OsJAZ1 is strongly up-regulated under drought stress, and its overexpression of OsJAZ1 negatively modulates the drought resistance [72]. Cis-acting regulatory elements in the promoter regions play a critical role in regulating gene expression by controlling the affinity of transcription factors. In this study, the cisacting elements related to phytohormones (MeJA, SA, IAA, ABA, GA, and ETH) and stress responses (wound, osmotic, and low temperature) were discovered in the putative promoter regions of the pineapple JAZ genes (Figure 4). The expression profiles of AcJAZs under phytohormone (MeJA, IAA, ABA, SA, and 6-BA) and abiotic (cold, heat, salt, and osmotic) treatments ( Figure 4) suggest that AcJAZ genes play a pivotal role in plant response to abiotic stresses.

Conclusions
In this study, 14 pineapple JAZ genes were identified and classified into five groups. The AcJAZ genes shared a similar gene structure and motif arrangement. The conservative motifs Jas and TIFY were present in all AcJAZs with their specialized functions. The putative promoter regions of AcJAZs contain between three and eight abiotic stress-responsive cis-acting elements. The expression patterns of AcJAZs in different tissues and in response to various abiotic stresses suggested that AcJAZs were associated with plant development and differentiation and significant response to different stresses and phytohormones. This finding indicates that AcJAZs may be involved in multiple biological processes during growth and development as well as resisting stresses. Moreover, the BiFC identification of protein interactions of AcJAZs with the central JA-signaling regulators suggested that AcJAZs and other vital players function in the JA-signaling pathway in response to abiotic stresses in pineapple. The results will be helpful in improving breeding for stress tolerance in pineapple.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biology11030445/s1, Figure S1. The alignment of 14 AcJAZ proteins showed the tify motif and Jas domain; Figure S2. Motif logos of AcJAZs; Figure S3. Motifs in AcJAZ identified by Pfam; Figure S4. The protein-protein interactions among central regulators of the JA-signaling pathway in pineapple (continued). Table S1. The list of RT-PCR Primers; Table S2. Prediction of subcellular localization of AcJAZ proteins; Table S3. Prediction of subcellular localization of OsJAZ proteins. Table S4. The cis-acting abiotic responsive elements in Pineapple JAZ genes; Table S5. MYC2 genes in pineapple; Table S6. JAM1 genes in pineapple; Table S7. NINJA genes in pineapple; Table S8. BiFC identification of protein-protein interactions among central regulators of the JA-signaling pathway in pineapple; Supplemental File S1. All protein sequences used for phylogenetic analysis in this study; Supplemental File S2. The motif information of pineapple JAZ proteins.