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Article

Genome-Wide Identification, Characterization, and Expression Pattern Analysis of the JAZ Gene Family in Wax Apple (Syzygium samarangense)

by
Liang Li
1,†,
Weijie Huang
1,2,†,
Limei Tang
1,2,
Ling Xu
1,
Yajun Tang
1,
Xiuqing Wei
1,* and
Jiahui Xu
1,*
1
Fruit Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China
2
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(10), 1011; https://doi.org/10.3390/horticulturae10101011
Submission received: 2 September 2024 / Revised: 19 September 2024 / Accepted: 20 September 2024 / Published: 24 September 2024
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Abstract

Wax apple (Syzygium samarangense) is a characteristic tropical fruit with great potential value, but the cold sensitivity and male sterility limit its cultivation and breeding. Despite the important role in JA-mediated cold-stress resistance and flower development, jasmonate ZIM-domain (JAZ) proteins have not been identified in wax apple. This study employed sequence blast, phylogenetic analysis, and RT-PCR to identify SsJAZs in wax apple systematically. First, 14 SsJAZs family members with TIFY and Jas domains were identified and named according to the distribution on ten chromosomes. Low-temperature responsiveness elements and TGACG-motif (response to MeJA) were abundant in the promoters of most SsJAZs. The transcription factors ERF and MYB were predicted to be involved in the regulation of SsJAZs. RT-PCR analyses showed that SsJAZs were mainly expressed in mature leaves and flowers. Further analysis revealed differences in the expression patterns of SsJAZs under cold treatment, as well as in different anther stages. SsJAZ proteins were predicted to interact with the N terminus of SsMYC2 protein via the Jas motif at the C-terminal domain. This study characterized the SsJAZs family members and examined the expression patterns in different samples. The results will advance our understanding of the role of SsJAZs in wax apple.

1. Introduction

Jasmonates (JAs), including jasmonic acid and the relational derivatives methyl cis-jasmonate (MeJA) and (+)-7-iso-jasmonoyl-L-isoleucine (JA-Ile), are a class of small plant signaling molecules that are essential for the survival of plants in nature. As vital endogenous plant hormones, they play a crucial regulatory role in plant responses to biotic and abiotic stresses [1]. JAs help improve the cold resistance of Arabidopsis thaliana [2] and rice [3,4] by activating C-repeat binding factor (CBF) protein through JA signal transduction. Due to its ability to easily penetrate cell membranes, MeJA is often used as an exogenous hormone for spraying to study the response mechanism of plants to JAs hormone stress. By being sprayed with MeJA, maize was induced to produce more toxic proteins that enhanced its defense mechanism against Ostrinia furnacalis [5]. In addition, JAs are involved in root growth, reproductive development, fruit ripening, and other plant development processes [6]. Recent studies have shown that JAs also mediate the biosynthesis of multiple secondary metabolites in medicinal plants [7,8].
In plants, the core JA signal transduction module consists of three parts: a group of jasmonate ZIM-domain (JAZ) proteins that function as transcriptional repressors; the JA receptor F-box Coronatine Insensitive 1 (COI1) protein that forms an SCF (Skip/Cullin/F-box) E3 ubiquitin ligase complex (SCFCOI1); and the transcription factors (MYC2, one of the key TFs) that orchestrate the expression of JA-related effector genes [9]. Commonly, the concentration of bioactive JA-Ile is maintained at a low level in plants. In this state, the JAZ protein recruits the adaptor protein Novel Interactor of JAZ (NINJA) and the co-repressor Topless (TPL) to form an effective closed-complex corepressor (JAZ-NINJA-TPL complex) and interact with and prevent MYC2 from activating JA signaling-responsive genes. After plants sense the stress, JA-Ile is synthesized rapidly and transferred to the nucleus. In the nucleus, the high concentration of JA-Ile is perceived by the SCFCOI1 complex and promotes the interaction of COI1 with JAZ proteins. Mediator subunit 25 (MED25) contributes to this stage. Then, a JA-Ile-dependent receptor complex is formed and leads to the ubiquitination (degradation) of JAZ proteins in the 26 S proteasome. Subsequently, the TF MYC2 and other TFs suppressed by the JAZ-NINJA-TPL complex regain transcriptional activity and finally activate the expression of JA signaling-responsive genes [6,10]. Accordingly, JAZ proteins were considered the critical negative regulator in the pathway of JA signaling.
The regulatory ability of JAZ proteins is attributed to two conserved domains: the TIFY domain (also named the ZIM domain) and the Jas domain (also named the CCT_2 domain) [11]. The highly conversed TIFY domain is located in the middle of JAZ protein sequences towards the N-terminal region. The TIFY domain has been confirmed to mediate the homomeric and heteromeric interactions of JAZ proteins [12]. Additionally, JAZs interact with NINJA protein via the TIFY domain to recruit TPL to form the JAZ-NINJA-TPL complex [13]. The Jas domain is located at the C-terminal of JAZs. JAZ proteins interact with MYC2 or other TFs through the Jas domain and repress their transcriptional activation activity [6]. The LPIARR motif in the N-terminal of the Jas domain was reported to play a critical role in the JA-Ile-dependent interaction with COI1 to destabilize the repressor [14]. More than one dozen JAZ-interacting TFs have been identified, indicating the functional diversification of JAZ proteins [12]. In Arabidopsis, the jaz2 mutant loses stomatal defense to disease [15], jaz4 jaz9 mutants delay flowering [16,17], and jaz6 jaz8 mutants enhance JA-regulated root growth inhibition and defense against the necrotrophic fungus [18]. Interestingly, lack of AtJAZ4,7,8 would accelerate dark-induced leaf senescence [19], while lack of AtJAZ6 appeared to delay it [20]. The high-order jaz mutant jaz1-2/2-3/3-4/4-1/5-1/6-4/7-1/9-4/10-1/13-1 (jazD) exhibits short roots, growth stunting, enhanced anthocyanin, and glucosinolate biosynthesis [21]. The undecuple mutant jaz1/2/3/4/5/6/7/9/10/11/12 (jaz1-7,9-12) is hypersensitive to JA in root growth inhibition and hook curvature, suggesting a redundant repression mechanism [22]. In Artemisia annua, AaJAZ8 protein suppressed artemisinin biosynthesis by AaTCP14-AaORA complex and MYC2, while AaJAZ9 protein positively regulated it [23,24]. MdJAZ1 and MdJAZ2 proteins negatively regulated JA-mediated cold tolerance in apple by interfering with the transcriptional activity of MdABI4 and a novel B-box (BBX) protein, MdBBX37 [25,26]. Exogenous MeJA induced the various physiological processes in plants. MeJA induced the expression of fifteen out of twenty CwJAZs and the accumulation of β-Elemene in Curcuma wenyujin [8]. The expression of three out of eighteen GbJAZs was reduced, and the accumulation of ginkgolides was induced in the fibrous root by MeJA treatment [7]. The response of JAZ family members to MeJA is diverse. The functional specificity and redundancy of JAZ proteins in plants still need further investigation.
Wax apple (Syzygium samarangense (Blume) Merr. and Perry), native to the Malay Archipelago, is a characteristic tropical fruit from the Myrtaceae family. Because of the crisp and juicy taste, the aroma of roses, low-acidity and low-sugary flavor, and richness in antioxidant compounds, wax apple has become an economically important fruit in Southeast Asia [27]. Wax apple tree exhibits hypersensitivity to cold stress tolerance, and the leaf mesophyll tissue of adult trees would be damaged under an environment of less than 7 °C [28]. The suitable cultivation area for wax apples is restricted to a few warm and humid regions. Therefore, it is significant to investigate key regulators in JA-mediated cold tolerance in wax apple. In addition, the seedless character of wax apple fruit is an important quality popular among consumers. Male sterility caused by anther dehiscent disorder is a key reason for the seedless fruit of the wax apple [29]. Through transcriptome analysis for different flowering stages in wax apple, we found several differentially expressed genes involved in JA biosynthesis and signaling pathways, such as JAR1 (a JA-Ile synthesizing enzyme), JAZs, and MYC2 (unpublished data). The role of JAZ proteins in regulating anther dehiscence deserves more attention and exploration. Hence, it is necessary to analyze the JAZ gene family of wax apple in depth, which has not been systematically identified.
According to the recently released high-quality genome [29], we can better study the members and function of the JAZ gene family in wax apple. In this study, the SsJAZ gene family of wax apple was systematically identified. Chromosomal location, gene structure and conserved domain, phylogenetic tree, and promoter analysis were performed to characterize this gene family. Previous studies have explored the responses of the JAZ gene family against cold, hormones, or other stimuli in some plants. The role of JAZ genes in anther dehiscence in Arabidopsis was also revealed. Cold stress sensitivity and anther dehiscence disorder are two major issues affecting wax apple breeding. To explore the function of the SsJAZ gene family in stress and anther dehiscence in wax apple, organ-specific, time-specific, MeJA-induced, and cold-induced expression profiles of SsJAZ genes were determined. Furthermore, the interaction between SsJAZs and other relative proteins was predicted by AlphaFold 3. These results enable further understanding of the biological functions of SsJAZs in wax apple.

2. Materials and Methods

2.1. Plant Materials and Treatment

The wax apple ‘Tub Ting Jiang’ (‘Tub’) and ‘DK No.3’ (‘DK’) accessions were chosen as plant materials in this study. The plant materials were maintained by the Fujian Academy of Agricultural Sciences in the wax apple GenBank Field (Yunxiao town, Zhangzhou city, Fujian province, China, coordinates: 23°59′26.95″ N; 117°16′57.39″ E). Six-year-old ‘Tub’ and ‘DK’ trees were selected as materials for organ-specific and time-specific expression analysis of related genes. For organ-specific expression analysis, different organs, including roots, stems, young leaves, mature leaves, flowers (at blooming stage), and fruits (at ripening stage), were collected from ‘Tub’. For time-specific expression analysis, the five stages of anther (namely T1 to T5) in two wax apple accessions ‘Tub’ and ‘DK’ were sampled according to a previous study [29].
Because of the small size and ease of whole plant treatment, three-year-old ‘Tub’ air-layering plants were selected and subjected to MeJA stress and cold treatment. As to MeJA treatment, the entire plants in soil pots were treated with MeJA at concentrations of 400 μM. The specific operation methods refer to the previous study [7]. We sprayed half of the MeJA solution evenly over the leaves and stems (above-ground parts of plants) and irrigated the rest evenly to the roots. The mature leaves were collected at 0, 0.5, 3, 6, 9, and 24 h after treatment. For cold treatment, the air-layering plants of ‘Tub’ were placed in cold storage (set at 7 °C), and the mature leaves were collected after 0 and 9 h after treatment [28]. Biological replicates were collected from three trees for each sample. All samples were separately collected and quick-frozen in liquid nitrogen and stored at −80 °C.

2.2. Identification of the JAZ Gene Family Members in Wax Apple

The genome files of wax apple were obtained from the National Genomics Data Center (under accession numbers GWHDUEN00000000 and GWHDVFZ00000000). The AtJAZ protein sequences of Arabidopsis thaliana were obtained from TAIR (https://www.arabidopsis.org/, accessed on 3 January 2024). First, we used AtJAZ protein sequences to search for potential SsJAZ proteins with the program “Blast Several Sequences to a Big Database” (E-value: 1 × 10−5) in TBtools-II [30]. Then, all putative protein sequences were further confirmed using Pfam [31] (https://www.ebi.ac.uk/interpro/search/sequence/, accessed on 6 January 2024) and NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi/, accessed on 6 January 2024). Only those sequences containing both TIFY (accession No. PF06200) and Jas (accession No. PF09425) conserved domains were selected. The chromosome location analyses and multiple amino acid sequence alignment of identified SsJAZ proteins were performed utilizing TBtools-II [30]. The proteins’ structural properties, including molecular weight and pI (predicted isoelectric), were analyzed by ExPASy-ProParam (https://web.expasy.org/protparam/, accessed on 30 January 2024). The subcellular localization of SsJAZ proteins was predicted by Plant-mPLoc th(http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 30 January 2024).

2.3. Gene Structure and Phylogenetic Analysis

The gene structure of each SsJAZs was constructed based on genome sequence and annotation files. The conserved domains were analyzed by Batch-Search in NCBI (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi/, accessed on 31 January 2024), and motifs were analyzed by MEME Suite (https://meme-suite.org/meme/tools/meme, accessed on 31 January 2024). The above results were visualized using the program “Gene Structure View (Advanced)” in TBtools-II [30].
The amino acid sequences of the JAZ gene family and annotation profiles of Citrus clementina, Eucalyptus grandis, Vitis vinifera, Corymbia citriodora, Populus trichocarpa, Prunus persica, and Malus domestica were obtained from the Phytozome website (https://phytozome-next.jgi.doe.gov/, accessed on 3 January 2024) (Table S1). A phylogenetic tree of JAZ proteins (SsJAZs, AtJAZs, CicleJAZs, EucgrJAZs, VvJAZs, CocitJAZs, PtrifJAZs, PrupeJAZs, and MdJAZs) was constructed using MEGA11 [32] with the maximum likelihood (ML) method. Then, the visualization of the evolutionary relationship tree was realized through TVBOT [33] (https://www.chiplot.online/tvbot.html, accessed on 31 January 2024). The collinearity analysis between wax apple and other species (Corymbia citriodora and Eucalyptus grandis) was conducted by using the program “One Step MCScanX” in TBtools-II [30].

2.4. Promoter Analysis

The upstream 2 kb sequence of the star codon for each SsJAZs was retrieved as putative promoters by using TBtools-II [30] based on the genomic sequences. Subsequently, cis-acting elements in the putative promoter region were predicted via PlantCARE [34] (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 6 February 2024) and visualized by TBtools-II [30]. Then, the potential transcription factors that bind to the promoter of SsJAZs were predicted by PlantRegMap [35] (https://plantregmap.gao-lab.org/regulation_prediction.php/, accessed on 10 July 2024) (p-value ≤ 10−6). The potential transcription factor regulatory network was shown by Cytoscape (v3.9.1) [36].

2.5. Quantitative Real-Time PCR (qRT-PCR) Analysis

Total RNA from each sample was extracted with an RNAprep Pure Plant Plus Kit (DP441, TIANGEN, Beijing, China). Then, the FastKing gDNA Dispelling RT SuperMIX Kit (KR118, TIANGEN, Beijing, China) was used for cDNA synthesis. The real-time qPCR was performed on the Roche LightCycler 480II system (Roch Basel, Switzerland) using a Tip Green qPCR SuperMix Kit (AQ141, TransGen, Beijing, China), according to the manufacturer’s instructions. The reaction program included initial denaturation at 94 °C for 30 s, followed by 45 cycles of denaturation at 94 °C for 5 s and annealing and extension at 60 °C for 30 s, and finally, melting curve detection at 65–95 °C. Each plate was repeated three times in independent runs for all genes. The transcript levels of genes were normalized to those of SsEF-1a (internal control gene) [37]. The relative expression levels of each gene were calculated by the 2−ΔΔCt method. We also checked the expression levels of several key genes (SsCOI1, SsMED25, and SsMYC2) in the JA signing pathway associated with the SsJAZ genes. The related primers were designed with Primer5 software and listed in Table S2.
The results were shown as the mean ± standard error of the mean. All statistical analyses were performed using IBM SPSS Statistics 26 software (International Business Machines Corp., Armonk, NY, USA). Independent two-sample t-tests (*, p < 0.05) were performed to compare two treatment groups. The significance difference of multiple groups of data was determined by one-factor ANOVA analysis (p < 0.05). The results were visualized by OriginPro 2021. The heatmap was constructed by TBtools-II [30].

2.6. Prediction of Protein Interaction

Protein interaction between SsJAZs and SsMYC2 proteins was predicted by AlphaFold 3 (https://www.alphafoldserver.com, accessed on 24 August 2024) [38]. The amino acid sequence of SsJAZs and SsMYC2 proteins was uploaded to the platform and predicted with the default configuration. Previous studies have confirmed a directed interaction between AaJAZ3 and AtMYC2 [6]. Therefore, we also uploaded the amino acid sequences of AaJAZ3 and AtMYC2 and predicted their interaction. After the prediction through AlphaFold 3, five models ranked based on the confidence score were outputted. The highest-ranked model was chosen for visualizing the structures and interacting residues between two proteins through the PyMOL molecular graphics system (v.2.6.0).

3. Results

3.1. Genome-Wide Identification of the JAZ Gene Family in Wax Apple

A total of 14 members of the JAZ gene family in wax apple were found by BLAST with Arabidopsis thaliana and Eucalyptus grandis, designated SsJAZ1-SsJAZ14 based on their chromosomal locations (Figure 1 and Table S3). The SsJAZs were unevenly distributed on 9 chromosomes. Sequence analysis showed that these SsJAZs encode proteins of 133 to 397 amino acids, with the corresponding molecular weight varying from 15.31 to 42.24 kDa. Through subcellular localization prediction, it was indicated that all SsJAZs were located in the nucleus. The alignment and conserved motif analysis showed that the Jas (PF09425) conserved domain was located at the C-terminal and the TIFY domain (PF06200) was located in the middle of sequences towards the N-terminal region of all SsJAZ proteins (Figure 2).

3.2. Gene Structure and Phylogenetic Analysis of SsJAZs

First, the 14 SsJAZ proteins were divided into 5 subfamilies by phylogenetic analysis (Figure 2). Then the UTR, CDS, and intron distribution of SsJAZs were analyzed. Members of the same subfamily exhibited similar patterns of exon and intron distribution, including exon length and intron number. It was found that SsJAZ5, SsJAZ8, and SsJAZ13 did not have a 5′ UTR structure. Based on the results of the online software MEME’s analysis, we predicted the other motifs of SsJAZs by finding 10 motifs, and each SsJAZs contained 3 to 6 motifs. There were a few motifs that were shared by all members, including motif1, motif2, and motif3. As well, some motifs were specific to the subfamily. The motif4 and motif7 were only found in subfamily V, and motif9 was unique to subfamily IV.
To explore the affinities of JAZ proteins in wax apple (14 SsJAZs), Arabidopsis thaliana (13 AtJAZs), Citrus clementina (6 CicleJAZs), Eucalyptus grandis (9 EucgrJAZs), Vitis vinifera (7 VvJAZs), Corymbia citriodora (12 CocitJAZs), Populus trichocarpa (7 PtrifJAZs), Prunus persica (7 PrupeJAZs), and Malus domestica (13 MdJAZs), a rootless phylogenetic tree was constructed with maximum likelihood (ML) method by MEGA 11 (Figure 3). All JAZ proteins were classified into five subgroups, named Group I to Group V. In each branch, it was observed that SsJAZs proteins tended to be on the same branch as different CocitJAZs and EucgrJAZs, indicating the close relationship between wax apple, Corymbia citriodora, and Eucalyptus grandis.
To further study the evolutionary relationship of JAZ genes between wax apple and other different species, a collinear map of wax apple with Corymbia citriodora and Eucalyptus grandis was constructed (Figure 4 and Table S4). We found that the 13 SsJAZs of wax apple have collinear relationships with 14 genes in Corymbia citriodora. In addition, there were 11 SsJAZs of wax apple that have collinear relationships with 13 genes in Eucalyptus grandis. The results showed that JAZ genes exhibited a strong synteny between wax apple, Corymbia citriodora, and Eucalyptus grandis. SsJAZ14 did not have co-localized genes with any of the three species, suggesting that SsJAZ14 may have formed after plant differentiation.

3.3. Cis-Acting Element Analysis of SsJAZs

In order to identify the potential biological functions of SsJAZs, the 2 kb regions upstream of the start codon of SsJAZs were extracted and analyzed with the PlantCare online website. The results showed that environmental stress-related elements and hormone-related elements were abundant in the promoter of SsJAZs (Figure 5 and Table S5). The environmental stress-related elements, including light responsive elements (G-box, GATA-motif), anaerobic inducible elements (ARE), low-temperature responsiveness elements (LTR), and drought-inducibility elements (MBS), were identified. It is notable that all of the above environmental stress-related elements were presented in the promoter of SsJAZ13, indicating the important role of SsJAZ13 in the response to various environmental stresses. Among all hormone-related elements, ABRE (response to ABA) and CGTCA-motif and TGACG-motif (response to MeJA) took up the major proportion in SsJAZs. ABA-responsive elements were present in the promoter regions of almost all SsJAZs, except for SsJAZ5. And MeJA-responsive elements appeared in almost all SsJAZs except for SsJAZ9 and SsJAZ10. Significantly, the promoters of the three SsJAZs (SsJAZ3, SsJAZ8, and SsJAZ11) were enriched for various hormone-related elements, including ABA-, MeJA-, Auxin-, Gibberellin-, and Salicylic acid-responsive elements. Accordingly, SsJAZs may be mainly involved in plant life activities by responding to environmental stress and hormone signal transduction.
Then, PlantRegMap (p-value ≤ 10−6) was used to predict the potential TFs that bind to the promoter of SsJAZs. The diverse TF families, including ERF, Dof, C2H2, MYB, and MIKC-type MADS, were predicted to be involved in the regulation of SsJAZs (Figure 6 and Table S6). Remarkably, the ERF TFs were the most abundant and mainly bound to the promoter of SsJAZ5, SsJAZ8, and SsJAZ13. Another abundant member was Dof TFs, which was mainly involved in the regulation of SsJAZ2, SsJAZ10, and SsJAZ11. In addition, 8 MYB TFs were predicted and bound to the promoter of SsJAZ4, SsJAZ5, SsJAZ6, and SsJAZ13. The MIKC-type MADS TFs, associated with plant growth and development, were only involved in the regulation of SsJAZ11.

3.4. Expression Patterns of SsJAZs

The expression patterns of SsJAZs were analyzed by RT-qPCR in different organs of wax apple, including roots, stems, young leaves, mature leaves, flowers, and fruits. Based on the results (Figure 7 and Table S7), we found that most of SsJAZs were expressed at high relative levels in mature leaf and flower organs, except for SsJAZ6. Among them, SsJAZ8, SsJAZ9, and SsJAZ13 kept the high expression levels in all organs. However, the overall expression level of SsJAZs in roots and stems. SsJAZ2 and SsJAZ4 showed no expression or extremely low expression levels in all organs.
The jasmonic acid signaling involved in cold tolerance responsive mechanisms in many plants. So, we analyzed the relative expression levels of SsJAZs under cold treatment (Figure 8 and Table S8). The results showed that the relative expression levels of SsJAZ1, SsJAZ2, SsJAZ4, and SsJAZ14 were up-regulated by cold treatment. While the expression of SsJAZ3, SsJAZ7, SsJAZ8, SsJAZ10, SsJAZ11, and SsJAZ13 was inhibited under low temperature.
Cis-acting element analysis revealed that the MeJA hormone-responsive element was present in the promoters of many SsJAZs. As a commonly used hormone treatment, MeJA treatment could more clearly explore the JAZ gene response in JA signal transduction. Then we conducted RT-qPCR to explore the response pattern of SsJAZs to MeJA (Figure 9 and Table S8). We found that the relative expression levels of 7 SsJAZs (SsJAZ5, SsJAZ6, SsJAZ7, SsJAZ8, SsJAZ10, SsJAZ11, and SsJAZ12) were up-regulated significantly under MeJA stress with different response times. The expression of SsJAZ11 was significantly increased at 0.5 h after MeJA treatment, which indicated that SsJAZ11 was sensitive to MeJA. The expression of SsJAZ8, SsJAZ10, and SsJAZ12 was significantly induced at 3 h after MeJA stress. And the expression levels of SsJAZ5, SsJAZ6, and SsJAZ7 were significantly up-regulated at 6 h after MeJA exposure. We noticed that the expression of SsJAZ1 and SsJAZ14 showed a trend of marked decreasing at 0.5 h and then increasing at 24 h after MeJA treatment.
In the previous study, we found that the wax apple ‘Tub’ exhibits abnormal dehiscence in anther [29]. Jasmonic acid signaling was reported to play an important role in anther dehiscence regulation. Therefore, we test the expression patterns of SsJAZs from anther organs at different stages in two wax apple varieties (‘Tub’ with anther abnormal dehiscence and ‘DK’ with anther normally dehiscence). Similar expression patterns were observed for all SsJAZs between the two wax apple varieties, but the expression difference fold was inconsistent (Figure 10 and Table S8). Almost all SsJAZs were actively transcribed (except for SsJAZ2 and SsJAZ4) at the T4 and T5 periods, which were critical stages for anther dehiscence. But most of the SsJAZs showed a higher expression difference fold in the variety ‘DK’ compared to the variety ‘Tub’. In the JA signaling pathway, COI1 engages with JAZ and facilitates its degradation in collaboration with MED25, resulting in the reactivation of MYC2 proteins. So, we checked the expression patterns of these genes directly related to the JAZ genes (Figure S1). Those relative regulatory genes showed the same expression patterns between two varieties and were up-regulated at the T4 period. The relative expression of SsCOI1 reached a maximum at the T4 period in variety ‘DK’, and the up-regulation multiple was more than 3 times. While the relative expression of SsCOI1 showed no significant difference between the T1 and T4 periods in the variety ‘Tub’. The relative expression level of SsMED25 at the T4 period was up-regulated by more than 8 times compared with that at the T1 period in variety ‘DK’. The up-regulation multiple of SsMED25 was only 1.3 times in the variety ‘Tub’. In a word, SsJAZs and its relative regulatory genes were up-regulated in the T4 and T5 periods of anther, and were more active in variety ‘DK’ than in variety ‘Tub’.

3.5. Interaction between SsJAZs and SsMYC2 Proteins

The interaction between JAZ and MYC2 proteins orchestrated the expression of JA-related effector genes. To further investigate the function of SsJAZ proteins in wax apple, the interaction between SsJAZs and SsMYC2 proteins was predicted by the Alphafold 3 platform (Figure 11 and Figure S2). We first predicted the interaction between AtJAZ3 and AtMYC2 proteins in Arabidopsis thaliana, and the result was consistent with the previous study [6], that is, the interacting residues were located at the C-terminal domain of the AtJAZ3 protein. This indicated that the Alphafold 3 platform could predict the JAZ protein interaction model effectively and accurately. Then, we performed the same method to predict the interaction between SsJAZs and SsMYC2 proteins in wax apple. Based on the confidence score (Table S9), SsJAZ2, SsJAZ7, and SsJAZ11 proteins showed a strong interaction relationship with SsMYC2. The interaction structures indicated that SsJAZs proteins were predicted to interact with the N terminus of the SsMYC2 protein. The interacting residues of SsJAZs proteins were distributed in the Jas motif at the C-terminal domain.

4. Discussion

4.1. The Identification of SsJAZ Gene Family

The JAZ proteins play crucial roles in many physiological metabolic processes in plants. The presence of both TIFY and Jas conserved domains is a distinctive feature of the JAZ gene family [39]. According to this standard, JAZ gene family members of Arabidopsis (13 AtJAZs) [22], Taxus media (12 TmJAZs) [40], Populus trichocarpa (7 PtrifJAZs) [41], Prunus persica (7 PrupeJAZs) [42], and Ginkgo biloba (18 GbJAZs) [7] were identified. Due to the large number of species and the lack of valid genomic data, few JAZ gene family numbers have been identified in the Myrtaceae family. Based on the recently released genome data of wax apple and conserved domain analyses, 14 SsJAZs from wax apple were identified in this study. The number of JAZ gene family members identified in wax apple was comparable to that in Arabidopsis. There are differences in the number of gene families in different species due to the occurrence of repeated events. Since a tandem duplication gene cluster containing nine JAZ genes has been found, tandem duplication was considered the main event in which birch expands members of the JAZ gene family in Ginkgo biloba [7]. In this study, 14 SsJAZs were distributed on 9 chromosomes, and only one tandem duplication gene cluster containing two JAZ genes (SsJAZ13 and SsJAZ14) was found in wax apple (Figure 1). Our results provide a further understanding of the evolutionary relationship of the JAZ gene family in the Myrtaceae family.
Through sequence alignment, we found a significant difference in the LPIARR motif of the Jas domain among the SsJAZ proteins. The Jas domain of SsJAZ1, SsJAZ8, SsJAZ9, and SsJAZ10 proteins contains the complete LPIARR motif. Some studies have shown that the LPIARR motif could seal JA-Ile on the COI1-JAZ interface. Proteins without the LPI motif can still make direct contact with JA-Ile but cannot fix it [43,44]. Those types of proteins could only interact with COI1 proteins at high concentrations of JA-Ile [45]. In addition, the Jas domain is also a key factor for JAZ interaction with the MYC2 protein. Truncated proteins containing only the Jas domain are sufficient for the interaction with the MYC2 protein but not with COI1 [46]. And the interaction between JAZ and MYC2 is independent of the hormone. We found that the interaction residues of SsJAZs and SsMYC2 proteins were mostly located in the Jas domain, and a few were located in the TIFY motif. (Figure S2). The interacting residues of SsJAZ and SsCOI1 proteins are not only located in the Jas and TIFY domains, but also a long fragment of residues was predicted to be located in the N terminus of JAZ protein (Figure S3). The way SsJAZ proteins interact with SsMYC2 and SsCOI1 proteins through different motif regulation in wax apple needs further exploration.

4.2. The Role of SsJAZs in JA-Induced Cold Tolerance

As it is extremely susceptible to cold damage, improving cold tolerance is one of the important tasks in wax apple breeding. It has been suggested that the JAZ gene family was widely involved in plant development and defense, including the mechanism in response to low-temperature stress [47]. In Arabidopsis, AtJAZ1 and AtJAZ4 repressed the transcription function of INDUCER OF CBF EXPRESSION 1 TF (ICE1), thereby negatively regulating the ICE-CBF/DREB1 signaling and cold stress response [48]. Anthocyanins protect plants from a variety of stresses, including cold stress. AtJAZ1,8,11 proteins interacted with bHLH factor TT8 and MYB member MYB75 of WD-repeat/bHLH/MYB complexes by the C-terminal Jas domain and affected their transcriptional function in JA-induced anthocyanin accumulation [49]. In apple, MdJAZ1 and MdJAZ2 proteins were reported to interfere with the BBX37-ICE1-CBF module by interacting with MdBBX37 protein, thereby negatively regulating JA-mediated cold stress resistance [26]. MdJAZ1 and MdJAZ2 proteins were also found to repress cold tolerance through the JAZ-ABI4-ICE1-CBF regulatory modulate [25]. In this study, the transcription levels of three SsJAZs (SsJAZ3, SsJAZ7, and SsJAZ13) were significantly down-regulated under cold stress, which were consistent with the transcription levels under MeJA treatment (Figure 8 and Figure 9). We could speculate that MeJA induced by cold stress promoted the degradation of SsJAZ3, SsJAZ7, and SsJAZ13. It is worth noting that SsJAZ1 expression is up-regulated under both cold stress and MeJA treatment. Some studies reported that most identified JAZ genes were significantly up-regulated by exogenous MeJA treatment, suggesting that JAZ plays an important role in JA signal transduction [7,40,50]. However, this appears to be contrary to the JAZ protein negatively regulating the JA signal transduction pathway. In this study, SsJAZ genes of wax apple showed a variety of expression patterns under MeJA treatment, similar to those in Aquilaria sinensis [12] and Prunus persica [42]. SsJAZ2 and SsJAZ4 showed different expression trends under cold stress and MeJA treatment. These results indicate that the SsJAZs gene family has complex and diverse regulatory patterns in response to different stress induction. This may also be caused by the difference in JAs content within the plants. The accumulation of JAs in plant cells under stress was within a controllable range, while exogenous MeJA treatment may lead to excessive JAs stress in plant cells. Therefore, the response of the JAZ gene family to moderate and excessive JAs hormone stimulation was different. In addition, the efficiency of converting MeJA into JA-Ile within plant cells is also a point of concern. Only bioactive JA-Ile can initiate JA signal transduction, and the response mechanism of plant cells to the remaining MeJA deserves further investigation. We checked the promoter cis-acting elements of SsJAZ genes with different expression patterns under cold stress. The results showed that there were 8 cis-acting elements (LAMP-element, Gap-box, AT-rich sequence, AAAC-motif, G-Box, TATC-box, 3-AF3 binding site, and AT-rich element) only distributed in the promoter sequences of genes up-regulated by cold stress (Figure S4). Most of these cis-acting elements belong to the light responsive element and the gibberellin-responsive element. Whether the light response pathway and gibberellin response pathway are involved in the promotion of SsJAZ genes under cold stress in wax apple is still not clear. Thus, further investigation of the function diversity and redundancy of SsJAZs in wax apple will help to better elucidate the complex JA signaling pathway.

4.3. The Role of SsJAZs in JA-Mediated Anther Dehiscence

There are many studies that have shown that JA pathway-related genes were involved in the regulation of anther dehiscence in Arabidopsis. Mutations of JA biosynthetic pathway genes, including Defective in Anther Dehiscence 1 (DAD1), Fatty Acid Desaturation (FAD), and Allene Oxide Synthase (AOS), lead to the anther dehiscent disorder [51]. Since these genes only affect JA biosynthesis, the corresponding mutants of these genes could be rescued by exogenous application of MeJA [52]. As a critical member of the SCFCOI1 complex in JA signaling, COI1 targets JAZ for degradation to release MYC2 and activate JA signaling-responsive genes. The AtCOI1 mutation of Arabidopsis exhibits non-anther dehiscence and male sterility, which cannot be rescued by exogenous application of MeJA. The ZmCOI mutant of Zea mays also showed the same phenotypes as the AtCOI1 mutation [51]. As the key repressor in the COI1-JAZ-MYC2 pathway, JAZ proteins undoubtedly play an essential role in the regulation of JA-mediated anther dehiscence. To explore the potential role of SsJAZs in the JA-mediated anther dehiscence process, we checked the expression patterns of all SsJAZs in the five anther stages of wax apple accessions ‘Tub’ and ‘DK’ (Figure 10). The expression level of most SsJAZs was significantly up-regulated during the T4 period, except for SsJAZ2 and SsJAZ4. At stage T5, the expression levels of SsJAZ1 and SsJAZ6 were induced, and the expression levels of SsJAZ3, SsJAZ5, SsJAZ8, SsJAZ9, SsJAZ10, SsJAZ11, SsJAZ12, and SsJAZ13 were inhibited. Further, we noticed that SsJAZ2 and SsJAZ7 in the anther of ‘Tub’ were opposite to those in ‘DK’ at T4 or T5 period. The expression of SsJAZ2 was significantly inhibited at stages T4 and T5 in ‘Tub’. In contrast, the expression level of SsJAZ2 in stages T4 and T5 was comparable to that in stage T1, and no suppression was observed. In the anther of ‘Tub’, the expression level of SsJAZ7 was induced at stage T5, exhibiting only 1.5 times higher than that at stage T1. The expression level of SsJAZ7 at stage T5 displayed 3-folds higher than that at stage T1 in ‘DK’. TaJAZ7, TaJAZ8, and TaJAZ12 were found to have different expression patterns in two wheat lines with different anther cracking phenotypes [53]. In the ZmCOI mutant of Zea mays, three ZmJAZ genes were up-regulated and ZmJAZ3, ZmCOI1d were down-regulated [51]. Those JAZ genes identified in maize and wheat were suggested to be involved in the regulation of the abnormal anther dehiscence. Therefore, we speculated that the expression fluctuation of SsJAZ2 and SsJAZ7 identified may be associated with JA-mediated anther dehiscent disorder in wax apple. Further verification is needed to determine whether the simultaneous high expression of SsJAZ2 and SsJAZ7 is necessary for the anther dehiscence in wax apple. Our previous experiments did not observe the phenomenon of MeJA treatment promoting the anther dehiscence in ‘Tub’. MeJA treatment activated the expression of SsJAZ7 while inhibiting the expression of SsJAZ2 in mature leaves. The expression of JAZ family genes may vary in different MeJA treatment sites and concentrations. It will be very interesting to conduct research on the period and concentration of MeJA treatment of anthers in the future. In addition, we also checked the expression level of SsCOI1, SsMED25, and SsMYC2 (Figure S1). The expression levels of SsCOI1 and SsMED25 were significantly higher in the T5 period than those in the T1 period in the anther of ‘DK’. While the expression of SsCOI1 and SsMED25 was significantly suppressed in the T5 period compared to the T1 period in the anther of ‘Tub’. In Arabidopsis, AtCOI1 was suggested to activate JA-mediated anther development relative genes by promoting AtJAZ1,8,11 degradation and releasing AtMYB21 and AtMYB24 [54]. The specific function of SsCOI1 and SsMED25 in the mechanism of anther cracking regulated by JA remains to be further explored.

5. Conclusions

Our study identified and analyzed the 14 SsJAZ gene family members in wax apple. SsJAZ proteins were predicted to interact with SsMYC2 and SsCOI1 proteins through the Jas domain. We investigated differential organ-specific and cold stress-responsive expression patterns of 14 SsJAZs in wax apple. We found that SsJAZ2 and SsJAZ4 may have complex and diverse regulatory patterns in response to different stress inductions. SsJAZ2, SsJAZ7, SsCOI1, and SsMED25 would likely participate in the regulation of JA-mediated anther dehiscent. The result contributed to the further understanding of the SsJAZ gene family in wax apple.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10101011/s1, Figure S1. The expression patterns of key regulatory genes involved in JA signaling in ‘Tub’ and ‘DK’ from anther at different stages. Figure S2. Diagram of predicted interaction between SsJAZs and SsMYC2 proteins. Figure S3. Prediction of protein interaction between JAZ and COI1 proteins. Figure S4. Venn map of cis-acting elements of SsJAZ genes with different expression patterns under cold stress. Table S1. Protein sequences of JAZs in 8 species. Table S2. Primers for each gene. Table S3. Characteristics of identified SsJAZ genes in wax apple. Table S4. One-to-one orthologous relationships between wax apple and 3 species. Table S5. Statistics of cis-acting elements in promoters of SsJAZ genes. Table S6. Regulation network between potential TFs and SsJAZ genes. Table S7. Relative expression profile of SsJAZs in different tissues (root, stem, young leaf, mature leaf, flower, and fruit) of wax apple. Table S8. Relative expression profile of SsJAZs under cold or MeJA treatment or anther at different stages. Table S9. The confidence score of predictive interaction models.

Author Contributions

Conceptualization, L.L.; methodology, L.L., W.H., L.T. and L.X.; software, L.L. and Y.T.; validation, L.L. and W.H.; investigation, L.L. and W.H.; resources, L.L. and L.X.; data curation, L.L.; writing—original draft preparation, L.L. and W.H.; writing—review and editing, J.X. and X.W.; supervision, X.W.; project administration, J.X. and X.W.; funding acquisition, L.L. and J.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the General Project of Fujian Natural Science Foundation (2023J01369), the High-quality Development beyond the “5511” Collaborative Innovation Project in Fujian province (XTCXGC2021016), and the Fundamental Scientific Research at Nonprofit Research Institutions in Fujian province (2021R10280010).

Data Availability Statement

Data are contained within the article and in the Supplementary Materials.

Acknowledgments

We appreciate all the people who have collaborated on this project.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chromosome distribution of SsJAZ gene family in wax apple genome. The colored rectangular bars represent the chromosomes of wax apple. The colored horizontal lines in rectangular bars represent the gene distribution density of chromosomes, and the red color represents higher gene density levels and blue color represents lower gene density levels.
Figure 1. Chromosome distribution of SsJAZ gene family in wax apple genome. The colored rectangular bars represent the chromosomes of wax apple. The colored horizontal lines in rectangular bars represent the gene distribution density of chromosomes, and the red color represents higher gene density levels and blue color represents lower gene density levels.
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Figure 2. Sequence analysis of SsJAZs in wax apple. Sequence logos and multiple sequence alignment of TIFY domains (A) and Jas domains (B) of SsJAZ proteins in wax apple. All SsJAZ proteins were classified into five subfamilies, named subfamily I to subfamily V. (C) Schematic diagrams of motif compositions of SsJAZ proteins in wax apple. Different motifs for SsJAZ proteins were indicated by different colored boxes. (D) Visualization of conserved domains of SsJAZ proteins in wax apple. (E) Visualization of gene structures for the SsJAZs.
Figure 2. Sequence analysis of SsJAZs in wax apple. Sequence logos and multiple sequence alignment of TIFY domains (A) and Jas domains (B) of SsJAZ proteins in wax apple. All SsJAZ proteins were classified into five subfamilies, named subfamily I to subfamily V. (C) Schematic diagrams of motif compositions of SsJAZ proteins in wax apple. Different motifs for SsJAZ proteins were indicated by different colored boxes. (D) Visualization of conserved domains of SsJAZ proteins in wax apple. (E) Visualization of gene structures for the SsJAZs.
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Figure 3. Unrooted phylogenetic tree of JAZ proteins from wax apple (SsJAZs), Arabidopsis thaliana (AtJAZs), Citrus clementina (CicleJAZs), Eucalyptus grandis (EucgrJAZs), Vitis vinifera (VvJAZs), Corymbia citriodora (CocitJAZs), Populus trichocarpa (PtrifJAZs), Prunus persica (PrupeJAZs), and Malus domestica (MdJAZs). Phylogenetic tree was constructed with the maximum likelihood (ML) method by MEGA 11 and visualized by the TVBOT web application [33].
Figure 3. Unrooted phylogenetic tree of JAZ proteins from wax apple (SsJAZs), Arabidopsis thaliana (AtJAZs), Citrus clementina (CicleJAZs), Eucalyptus grandis (EucgrJAZs), Vitis vinifera (VvJAZs), Corymbia citriodora (CocitJAZs), Populus trichocarpa (PtrifJAZs), Prunus persica (PrupeJAZs), and Malus domestica (MdJAZs). Phylogenetic tree was constructed with the maximum likelihood (ML) method by MEGA 11 and visualized by the TVBOT web application [33].
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Figure 4. Collinear analyses of JAZ genes between wax apple and three plants (Corymbia citriodora and Eucalyptus grandis). The gray lines represent collinear blocks in wide regions of the genomes, while the red lines show the orthologous relationship of JAZ genes.
Figure 4. Collinear analyses of JAZ genes between wax apple and three plants (Corymbia citriodora and Eucalyptus grandis). The gray lines represent collinear blocks in wide regions of the genomes, while the red lines show the orthologous relationship of JAZ genes.
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Figure 5. Prediction of cis-acting elements in the promoter of SsJAZs. (A) The number of cis-acting elements detected in each SsJAZs promoter. All elements were divided into five types, including growth and development relative elements, environmental stress-related elements, hormone-related elements, promoter-related elements, and others. (B) Visualization of cis-acting elements in the promoter of SsJAZs. (C) Venn map of hormone-related elements and environmental stress-related elements.
Figure 5. Prediction of cis-acting elements in the promoter of SsJAZs. (A) The number of cis-acting elements detected in each SsJAZs promoter. All elements were divided into five types, including growth and development relative elements, environmental stress-related elements, hormone-related elements, promoter-related elements, and others. (B) Visualization of cis-acting elements in the promoter of SsJAZs. (C) Venn map of hormone-related elements and environmental stress-related elements.
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Figure 6. Potential regulatory network of SsJAZs and potential TFs. The network was predicted by PlantRegMap and visualized with the Cytoscape (v.3.9.1). The orange box represents the SsJAZs. The ellipse with different colors represents TFs; light red shows ERF, violet shows Dof, purple shows MIKC-type MADS, green shows C2H2, light blue shows MYB, and light gray shows others.
Figure 6. Potential regulatory network of SsJAZs and potential TFs. The network was predicted by PlantRegMap and visualized with the Cytoscape (v.3.9.1). The orange box represents the SsJAZs. The ellipse with different colors represents TFs; light red shows ERF, violet shows Dof, purple shows MIKC-type MADS, green shows C2H2, light blue shows MYB, and light gray shows others.
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Figure 7. Expression profile of SsJAZs in different organs (root, stem, young leaf, mature leaf, flower, and fruit) of wax apple. Three biological replicates were analyzed. The expression heatmap was placed at the bottom right. The heatmap was drawn by TBtools, and red represents high expression level and green represents low expression level.
Figure 7. Expression profile of SsJAZs in different organs (root, stem, young leaf, mature leaf, flower, and fruit) of wax apple. Three biological replicates were analyzed. The expression heatmap was placed at the bottom right. The heatmap was drawn by TBtools, and red represents high expression level and green represents low expression level.
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Figure 8. Relative expression levels of SsJAZs after cold treatment. The * indicates significant differences by t-test (p < 0.05).
Figure 8. Relative expression levels of SsJAZs after cold treatment. The * indicates significant differences by t-test (p < 0.05).
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Figure 9. Relative expression levels of SsJAZs under MeJA treatment in 0, 0.5, 3, 6, 9, and 24 h. (A) Histogram of expression patterns of 14 SsJAZs under MeJA treatment. (B) The expression heat map of 14 SsJAZs under MeJA treatment. The different letters indicate significant differences by one-factor ANOVA analysis (p < 0.05).
Figure 9. Relative expression levels of SsJAZs under MeJA treatment in 0, 0.5, 3, 6, 9, and 24 h. (A) Histogram of expression patterns of 14 SsJAZs under MeJA treatment. (B) The expression heat map of 14 SsJAZs under MeJA treatment. The different letters indicate significant differences by one-factor ANOVA analysis (p < 0.05).
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Figure 10. The expression patterns of SsJAZs in ‘Tub’ and ‘DK’ from anther at different stages. The anthers of variety ‘Tub’ exhibit abnormal dehiscence. The anthers of variety ‘DK’ are rich in pollen and exhibit normal dehiscence. In each line chart, different letters indicate significantly different datasets based on one-factor ANOVA analysis (p < 0.05).
Figure 10. The expression patterns of SsJAZs in ‘Tub’ and ‘DK’ from anther at different stages. The anthers of variety ‘Tub’ exhibit abnormal dehiscence. The anthers of variety ‘DK’ are rich in pollen and exhibit normal dehiscence. In each line chart, different letters indicate significantly different datasets based on one-factor ANOVA analysis (p < 0.05).
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Figure 11. Prediction of protein interaction. (A) Close-up view of the interactions between the C-terminal of SsJAZ2 and the N-terminal of SsMYC2. The SsJAZ2-SsMYC2 interaction structure was shown in cartoon representation, interacting residues were shown in stick representation with wheat color, “KRKXRL” conserved interacting residues of SsJAZ2 were shown in stick representation with green color and labeled, and hydrogen bonds were shown as dashed lines. (B) Diagram of predicted interaction between AtJAZ3 and AtMYC2 proteins. (C) Diagram of predicted interaction between SsJAZ2 and SsMYC2 proteins. The amino acid sequences marked in red indicate interacting residues.
Figure 11. Prediction of protein interaction. (A) Close-up view of the interactions between the C-terminal of SsJAZ2 and the N-terminal of SsMYC2. The SsJAZ2-SsMYC2 interaction structure was shown in cartoon representation, interacting residues were shown in stick representation with wheat color, “KRKXRL” conserved interacting residues of SsJAZ2 were shown in stick representation with green color and labeled, and hydrogen bonds were shown as dashed lines. (B) Diagram of predicted interaction between AtJAZ3 and AtMYC2 proteins. (C) Diagram of predicted interaction between SsJAZ2 and SsMYC2 proteins. The amino acid sequences marked in red indicate interacting residues.
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MDPI and ACS Style

Li, L.; Huang, W.; Tang, L.; Xu, L.; Tang, Y.; Wei, X.; Xu, J. Genome-Wide Identification, Characterization, and Expression Pattern Analysis of the JAZ Gene Family in Wax Apple (Syzygium samarangense). Horticulturae 2024, 10, 1011. https://doi.org/10.3390/horticulturae10101011

AMA Style

Li L, Huang W, Tang L, Xu L, Tang Y, Wei X, Xu J. Genome-Wide Identification, Characterization, and Expression Pattern Analysis of the JAZ Gene Family in Wax Apple (Syzygium samarangense). Horticulturae. 2024; 10(10):1011. https://doi.org/10.3390/horticulturae10101011

Chicago/Turabian Style

Li, Liang, Weijie Huang, Limei Tang, Ling Xu, Yajun Tang, Xiuqing Wei, and Jiahui Xu. 2024. "Genome-Wide Identification, Characterization, and Expression Pattern Analysis of the JAZ Gene Family in Wax Apple (Syzygium samarangense)" Horticulturae 10, no. 10: 1011. https://doi.org/10.3390/horticulturae10101011

APA Style

Li, L., Huang, W., Tang, L., Xu, L., Tang, Y., Wei, X., & Xu, J. (2024). Genome-Wide Identification, Characterization, and Expression Pattern Analysis of the JAZ Gene Family in Wax Apple (Syzygium samarangense). Horticulturae, 10(10), 1011. https://doi.org/10.3390/horticulturae10101011

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