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Article

The SlJMJ15, a Putative Histone Demethylase Gene, Acts as a Negative Regulator of Drought Tolerance in Tomato

by
Lang Wu
*,
Hanling Zhao
,
Jiajia Xu
,
Fasen Lin
,
Qingxia Yan
,
Yan Liang
,
Danyang Xu
,
Yu Pan
,
Xingguo Zhang
and
Jinhua Li
*
Key Laboratory of Agricultural Biosafety and Green Production of Upper Yangtze River (Ministry of Education), College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(10), 1148; https://doi.org/10.3390/horticulturae11101148
Submission received: 26 August 2025 / Revised: 16 September 2025 / Accepted: 19 September 2025 / Published: 23 September 2025
(This article belongs to the Special Issue Breeding by Design: Advances in Vegetables)
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Abstract

JmjC domain proteins play crucial roles in plant growth and development, regulation of epigenetic processes, flowering control, and stress defence. However, these proteins have not been systematically identified or characterised in tomato. Here, we performed a genome-wide identification of JmjC domain-containing genes (JMJ family) in tomato and identified 23 SlJMJ genes within the tomato genome. Expression analysis indicated that SlJMJ15 was responsive to drought stress, prompting us to investigate its functional role in tomato plants. We found that SlJMJ15-RNAi lines displayed a severe dwarf phenotype, whereas SlJMJ15-overexpression lines exhibited increased drought sensitivity compared to wild-type plants, indicating that SlJMJ15 negatively regulates drought tolerance in tomatoes. Further investigation suggests that SlJMJ15 may reduce drought tolerance in tomatoes by modulating the expression of key genes involved in abscisic acid signalling pathways through its demethylation activity. This study deepens our understanding of the roles of SlJMJ family genes in tomato growth and abiotic stress responses, laying the foundation for developing strategies to improve drought stress tolerance in tomatoes.

1. Introduction

Epigenetic modifications are key regulatory mechanisms that enable plants to adapt to stress. Plants can precisely regulate stress-responsive genes without altering their genome sequences by dynamically modulating epigenetic patterns, including DNA methylation, non-coding RNAs, and histone modifications [1,2,3]. Histone modification regulates the abscisic acid (ABA) signalling pathway and genes associated with stomatal movement by modulating chromatin accessibility, which directly influences plant water-use efficiency and osmotic regulatory capacity [4,5,6]. Analysis of the epigenetic regulatory network mediated by histone modifications in drought tolerance can provide new perspectives for understanding the molecular mechanisms underlying plant environmental adaptability.
Histone methylation occurs on lysine and arginine residues within the N-terminal tails of histones, exerting either positive or negative effects on the activity of the associated DNA or genes [7,8]. For example, H3K9me2 methylation suppresses repetitive gene expression in plants, whereas H3K4, H3K36, and H3K79 methylation are associated with transcriptional activation. Histone methylation can be reversed by histone demethylases [6]. The histone demethylases, characterised by the presence of the jumonji C (JmjC) domain, catalyses histone lysine/arginine demethylation through Fe(II)- and α-ketoglutaric acid (α-KG)-dependent oxidative reactions [9,10].
The JmjC domain-containing proteins regulate plant growth, development, epigenetic processes, flowering, and stress defence [11]. For instance, AtJMJ12/REF6 promotes flowering, whereas AtJMJ11/ELF6 inhibits it [12,13]. AtJMJ15 overexpression (OE) enhances salt tolerance in Arabidopsis, whereas its deletion reduces salt tolerance [14]; OsJMJ703 affects stalk elongation [3]; and OsJMJ706 affects spikelet development, flower morphology, and floral organ number [15]. In alfalfa, MtJMJC5 not only regulates the circadian clock but also induces cold-responsive alternative splicing, serving as a protective mechanism against cold damage [16]. There is specificity for the action sites of JmjC proteins across different subfamilies. Mutations in the sites that bind divalent ferrous ions, α-ketoglutarate, and histone polypeptides in the JmjC domain can significantly affect catalytic activity [17]. Comparative analysis of Arabidopsis, rice, and human homologs has suggested that six or seven members lack demethylase activity [18]. RcJMJ40 is presumed to lack histone demethylase activity due to the absence of two divalent ferric ion-binding sites and one α-ketoglutarate-binding site [19]. Therefore, identification and characterisation of the JMJ gene family in various plants is particularly important.
JMJ gene family members have been identified in many plants, including Arabidopsis [18], rice [18], Zea [20], rose [19], and alfalfa [16]. Particularly, 21, 20, and 18 members of this family have been identified in Arabidopsis, rice, and sorghum, respectively [18]. In Arabidopsis, the JMJ protein family is classified into five subfamilies according to protein domain composition and catalytic specificity [18,21]. Among them, KDM5/JARID1 can reverse the methylation of H3K4me1/2/3 [22]; KDM4/JHDM3 can reverse the methylation of H3K9me2/3 and H3K36me2/3 [23]; KDM3/JHDM2 can reverse the methylation of H3K9me1 and H3K9me2 [23]; JMJD6 can reverse the methylation of H3K27me2/3 [24]; and proteins belonging to the JmjC domain-only subfamily can reverse H3K27me2/me3 methylation [25].
Most studies on the functional analysis of plant JMJ gene family members have primarily focused on Arabidopsis, rice, maize, and wheat; however, investigations into this gene family in tomatoes remain limited. The JMJ gene family is involved in tomato growth, development, and stress defence [26,27,28]. Specifically, SlJMJ6 (designated SlJMJ03 in this study; Solyc06g008490) promotes fruit ripening by activating the expression of ripening-related genes through H3K27me3 regulation [26]. Additionally, SlJMJ7 (SlJMJ01; Solyc02g069740) negatively regulates the disease resistance response of tomato to Pst DC3000 by reducing the expression of the salicylic acid-related genes SlPTI5 and SlWRKY28 [27]. In Arabidopsis, the ectopic SlJMJ524 expression (SlJMJ15; Solyc01g006680) confers transgenic plants with enhanced tolerance to cadmium stress [28]. Despite these findings, research on the roles of JMJ genes in the tomato drought stress response remains scarce.
Drought stress is one of the most significant abiotic factors limiting plant growth and development. Tomato, a highly drought-sensitive cash crop, has experienced yield reductions ranging from 30% to 50% under drought conditions, leading to direct economic losses exceeding $12 billion [29]. Therefore, this study aimed to identify and characterise the SlJMJ gene family in tomatoes using bioinformatics approaches and to analyse their roles in the drought stress response. Through expression analysis, we identified SlJMJ15 (also known as SlJMJ524 [28]; Solyc01g006680) from them as being involved in the drought stress response. Consequently, further investigation of drought tolerance was carried out using SlJMJ15 RNA interference (RNAi) and OE transgenic lines. Additionally, ABA and GSK-J1 (an H3K27me2/me3 demethylase inhibitor) treatments were performed to explore the regulatory mechanisms underlying SlJMJ15 activity. These findings provide valuable insights into the roles of SlJMJ family genes in tomato growth and abiotic stress responses, thereby contributing to strategies for improving stress tolerance in tomatoes.

2. Materials and Methods

2.1. Identification of SlJMJ Genes in Tomatoes

The JMJ protein sequences of Arabidopsis and rice that have been functionally characterised in previous studies were obtained from NCBI (http://www.ncbi.nlm.nih.gov; accessed on 6 April 2022) [18,30]. The Hidden Markov Model profile of the JMJ domain (PF02373) was downloaded from the Pfam database (http://pfam.xfam.org/; accessed on 26 September 2023) and used to identify SlJMJ genes from the tomato genome database ITAG 2.4 (https://solgenomics.net/; accessed on 26 September 2023) using Hmmsearch V3.1 [31] and Basic Local Alignment Search Tool algorithms (BLASTP) with an e-value threshold of <1 × 10−5 [32,33]. Moreover, redundant sequences in the same chromosomal location and short proteins (100 amino acids) were removed based on the physical localisation of all candidate genes. InterPro (http://www.ebi.ac.uk/interpro/; accessed on 6 October 2023) [34] and SMART (http://smart.embl-heidelberg.de/; accessed on 6 October 2023) [35] databases were used to verify the presence of the JMJ domain in all candidates. The identified candidate protein sequences containing the JMJ domain were the products of tomato SlJMJ genes and were assessed in this study. Finally, the physicochemical properties of the protein sequences were calculated using ExPASy (https://www.expasy.org/; accessed on 7 October 2023) [36], and the subcellular localisation of the SlJMJ gene family members was identified using WoLF PSORT (https://wolfpsort.hgc.jp/; accessed on 7 October 2023) [37].

2.2. Phylogenetic Analysis

JMJ protein sequences, including those encoded by SlJMJs, OsJMJs, and AtJMJs from tomato, rice, and Arabidopsis, were aligned using the MUSCLE algorithm [38] with default parameters in MEGA 7.0 [39]. A Model Generator programme was utilised to identify the most appropriate model for examining the evolutionary dynamics of the JMJ family of proteins. An unrooted neighbour-joining phylogenetic tree was constructed through comprehensive multiple sequence alignments of the JMJ proteins using MEGA 7.0. The members of the JMJ family were subsequently categorised into distinct classes based on the phylogenetic tree’s topology. The parameters employed in this analysis were as follows: pairwise deletion, Poisson’s model, and 1000 bootstrap replications.

2.3. Chromosomal Location, Gene Structure, and Cis-Element Analyses

Information on the physical location of SlJMJs on each chromosome was acquired from the genome annotation database of ITAG 2.4. The isodose distribution of SlJMJ genes was plotted using the MG2C software (http://mg2c.iask.in/mg2c_v2.1/; accessed on 10 October 2023) [40]. The exon and intron structures were graphically visualised using GSDS 2.0 (http://gsds.cbi.pku.edu.cn; accessed on 10 October 2023) [41]. The two genes of tandem repeat gene pairs must meet the conditions in which the identity exceeds 75% and the alignment length exceeds 75% of the longer sequence. Circos was used to represent the collinearity relationship [42]. The cis-elements of the promoter sequences (1.5 kb upstream of 5′ UTR) of SlJMJ genes were identified using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 10 October 2023) [43].

2.4. Expression Analysis and Validation

SlJMJ transcriptome data were obtained from the Tomato Functional Genomics Database (http://ted.bti.cornell.edu/; accessed on 7 October 2023) [44]. The data included reads from Illumina RNA-seq analysis of the roots, leaves, flower buds, and fruits at different growth stages of the tomato cultivar LA1585. The gene expression level was calculated as reads per kilobase per million for each tissue, normalised to Log2 (Supplementary Table S1), and visualised with heat maps in TBtools (Toolbox for Biologists) V0.49991 [45].
To analyse expression patterns under drought and salt stress conditions, tomato M82 (Solanum lycopersicum M82) plants were subjected to drought and salt stress treatments simulated using 20% PEG and 200mM NaCl, respectively. Surface-sterilised tomato seeds were sown on 1/2 Murashige–Skoog (MS) medium plates for germination. After 7 d, uniformly sized seedlings were transplanted onto fresh 1/2 MS medium plates. For the NaCl treatment, the seedlings were transferred to 1/2 MS medium plates supplemented with 200 mM NaCl and incubated for 0, 0.25, 0.5, 2, 12, or 24 h before sampling. For the PEG treatment, seedlings were transferred to 1/2 MS medium plates containing 20% PEG 6000 (equivalent to 200 mM osmotic potential) and incubated for 0, 0.25, 0.5, 2, 12, or 24 h before sampling. This method was adapted from a previous study [46]. In the same treatment, three plants were selected for each material at each time point (0, 0.25, 0.5, 2, 12, and 24 h), and the leaves of each plant were collected and immediately stored in liquid nitrogen. Following leaf sampling and total RNA extraction, the expression levels of SlJMJ03, SlJMJ08, SlJMJ15, SlJMJ20, SlJMJ21, and SlJMJ23 were determined using reverse transcription-quantitative PCR (RT-qPCR) (refer to Section 2.7). All data represent the averages of three biological and three technical replicates. The primers used for qRT-PCR are listed in Supplementary Table S2-1.

2.5. Construction of Plasmids, RNA Interference, and Plant Transformation

To investigate the function of SlJMJ15, RNAi and transgenic OE experiments were performed. SlJMJ15-OE transgenic lines and RNAi-mediated gene silencing lines were generated using the cultivar S. lycopersicum M82, as previously reported [47]. Supplementary Table S2-1 lists the primers used to construct these vectors. For RNAi vector construction, a 357 bp fragment from the SlJMJ15 coding sequence was amplified using gene-specific primers with 5′-attB1 and 5′-attB2 extensions added to the forward and reverse primers, respectively (Supplementary Table S2-1; 5′-attB1 and 5′-attB2 extensions are underlined). Subsequently, a recombination reaction was carried out between the PCR product and pHellsgate 2 vector (Invitrogen, Carlsbad, CA, USA) using BP clonase (Invitrogen), per the manufacturer’s instructions [48]. Two representative lines, RNAi2 (20 plants) and RNAi3 (20 plants), were selected for phenotypic analysis.
The plasmid used for SlJMJ15 OE was constructed in our laboratory using pMV, a vector modified from pBI121. Gene-specific primers for SlJMJ15-OE (Supplementary Table S2-1) were designed based on the cDNA sequence of SlJMJ15 (Soly01g006680, http://solgenomics.net/; accessed on 26 October 2023). The coding sequence of SlJMJ15 was amplified from M82 cDNA using PCR. Transgenic M82 tomato plants were generated through Agrobacterium tumefacien-mediated transformation (strain EHA105), as described previously [49]. PCR was used to detect the transgenes in genetically modified tomatoes using specific primers (Supplementary Table S2-1). Over 30 SlJMJ15 OE lines were identified. SlJMJ15 expression was analysed in eight selected lines. Two representative lines, OE8 (20 plants) and OE10 (20 plants), were selected for phenotypic analysis.

2.6. Soil Drought, Exogenous ABA, and GSK-J1 Treatments

2.6.1. Soil Drought Treatment

Surface-sterilised tomato seeds were individually sown in nutrient bowls (15 cm in diameter), which were filled with an equal weight of soil. All plants were cultivated in a growth chamber under a 16 h light (25 °C), 8 h dark (22 °C) cycle, and 80% relative humidity. Healthy seedlings with uniform growth patterns were divided into control and test groups. The control group was watered normally as the blank control, whereas watering of the test group was terminated to simulate soil drought conditions. The 1st day of drought treatment was defined as the day when the soil in the test group became visibly dry, followed by continuous observations. At 4 d after drought treatment, when a distinct phenotypic difference emerged between the two groups, samples from both groups were collected to determine relative electrical conductivity (REC), peroxidase activity (POD), catalase activity (CAT), and proline content (Pro). Three biological replicates were analysed for each sample. REC, POD activity, CAT activity, and Pro content were measured, as previously described [46]. Moreover, leaves from each group were stained with 3,3′-diaminobenzidine (DAB) and nitrotetrazolium blue chloride (NBT) to evaluate the extent of plant damage, as previously described [50,51]. When all plants exhibited severe wilting, a re-watering treatment was initiated, and plant recovery was monitored over 3–7 d.

2.6.2. ABA Treatment

Surface-sterilised seeds were sown on 1/2 MS medium plates for germination. Seeds of uniform size at the budding stage grown on 1/2 MS medium were transferred to 1/2 MS medium supplemented with 250 μM ABA (2 μMol/L) and incubated for 8 d. Subsequently, root length and plant height were measured for each plant. All root length and plant height data represent the averages of three biological and three technical replicates.

2.6.3. GSK-J1 Treatment

Surface-sterilised seeds were placed on a moist gauze in a Petri dish for germination. The budding seeds were divided into control and test groups and individually planted in cave discs containing 50 wells. The test group was treated with 100 μM GSK-J1 (an inhibitor of H3K27me2/me3 demethylases; solvent was 10% dimethyl sulfoxide) at 3, 7, 11, and 15 d post-germination [52]. The control group was treated with 10% dimethyl sulfoxide. The soil drought treatment was then immediately applied (2.6.1), and the status of the seedlings was observed daily. At 4 d after soil drought treatment, the plant height and drought tolerance level of each plant were measured. The drought tolerance level indicates the leaf growth status of plants 4 d after drought treatment and is categorised as follows: 1, more than three completely dead leaves; 2, two completely dead leaves; 3, one completely dead leaf with yellowing and wilting; and 4, no completely dead leaves and a small area with yellowing or wilting. All data represent the averages of fifteen biological and three technical replicates.

2.7. RNA Isolation and Reverse Transcription-Quantitative PCR Assays of ABA-Related Genes

At 4 d after drought treatment, three plants of each genotype (wild type [WT], OE8, and OE10) were selected, and the leaves from each plant were collected and stored in liquid nitrogen. Total RNA was extracted using an RNeasy Kit (Qiagen, Hilden, Germany) and treated with RNase-free DNase, according to the manufacturer’s instructions. The PrimeScript RT Reagent Kit (Takara Bio Inc., Kusatsu City, Japan) was used to synthesise cDNA using DNA-free total RNA as a template. For RT-qPCR, SYBR Premix Ex Taq (Takara Bio Inc.) was used on a Bio-Rad CFX96 instrument (Bio-Rad, Shanghai, China). PCR was performed in a 96-well iCycler. Eleven ABA-related genes, SlNCED3, SlAO1, SlAO2, SlPYL4, SlPYL7, SlPYL11, SlPP2C2, SlABI3, SlABI5, SlAREB1, and SlTAS14, were identified using RT-qPCR analysis. The specificity of the amplified fragments was verified by determining the melting temperature of the PCR products. Supplementary Table S2-1 lists the sequences of the primers used for RT-qPCR. Data were analysed using the 2−∆∆CT method, with SlActin (Solyc11g005330) as the reference gene [53]. All RT-qPCR data represent the averages of three biological and three technical replicates.

2.8. Statistical Analyses

All statistical analyses were performed using GraphPad Prism version 10 (GraphPad Software, San Diego, CA, USA), which combines statistical calculations with data visualisation for better reproducibility. Continuous variables with a normal distribution were presented as mean ± standard deviation (SD). One-way ANOVA was used to compare multiple groups, followed by Tukey’s post hoc test for pairwise comparisons. Four statistical significance levels were set at 0.01 ≤ p < 0.05, 0.005 ≤ p < 0.01, 0.001 ≤ p < 0.005, p ≤ 0.001, p < 0.05, respectively. GraphPad Prism automatically generated adjusted p-values for multiple comparisons with 95% confidence intervals.

3. Results

3.1. Identification, Physicochemical Characterisation, and Phylogeny Analysis of Tomato JMJ Gene Family

Based on the conserved amino acid sequence of the JmjC domain, we performed a comprehensive search and alignment using multiple public databases, ultimately identifying 23 SlJMJ gene family members in the tomato genome. These members were sequentially named SlJMJ01–SlJMJ23, based on their relative phylogenetic relationships (Supplementary Figure S1). The SlJMJ genes were unevenly distributed across the tomato genome (Supplementary Figure S2). Specifically, they were primarily located on chromosomes 1 (one member), 2 (six members), 3 (two members), 4 (five members), 6 (one member), 8 (five members), 9 (two members), and 10 (one member), whereas the remaining four chromosomes had no SlJMJ genes.
Physicochemical property analysis of the proteins encoded by the SlJMJ gene family revealed that the amino acid lengths range from 109 aa (SlJMJ16) to 1839 aa (SlJMJ04), the isoelectric points vary from 4.95 (SlJMJ10) to 9.51 (SlJMJ16), and the molecular weights span from 12.94 kDa (SlJMJ16) to 210.11 kDa (SlJMJ04). Bioinformatics analysis of subcellular localisation revealed distinct distribution patterns among JMJ family members. The majority (18 members, 78.26%) were predicted to localise to the nucleus, whereas cytoplasmic localisation was predicted for two members (SlJMJ11 and SlJMJ15, 8.69%). Organelle-specific localisation was observed in three members: SlJMJ07 (4.34%), in chloroplasts, and SlJMJ09 and SlJMJ21 (8.69%), both in mitochondria (Supplementary Table S3).
The amino acid sequences of SlJMJs, AtJMJs, and OsJMJs were subjected to multiple sequence alignment and subsequently plotted in a phylogenetic tree (Figure 1A). The analysis revealed that the 23 SlJMJ proteins could be classified into five subfamilies, KDM3, KDM4, KDM5, JMJD6, and JmjC-only, based on the classification system of Arabidopsis [18]. Specifically, the KDM3 subfamily comprises eight members (34.78%; SlJMJ16, 17, 18, 19, 20, 21, 22, and 23); the KDM4 subfamily includes five members (21.74%; SlJMJ05, 06, 07, 08, and 09); the KDM5 subfamily consists of four members (17.39%; SlJMJ01, 02, 03, and 04); the JMJD6 subfamily contains two members (8.70%; SlJMJ13 and SlJMJ14); and the JmjC-only subfamily includes four members (17.39%; SlJMJ10, 11, 12, and 15) (Supplementary Tables S3 and S4). Notably, SlJMJ02, SlJMJ04, SlJMJ05, SlJMJ06, SlJMJ10, SlJMJ11, SlJMJ12, SlJMJ13, SlJMJ14, SlJMJ21, SlJMJ22, and SlJMJ23 were closely related to AtJMJ16, AtJMJ17, AtJMJ12, AtJMJ11, AtJMJ32, OsJMJ709, AtJMJ31, OsJMJ711, AtJMJ22, AtJMJ27, AtJMJ24, and AtJMJ28, respectively. Furthermore, one pair of tandem duplications and one pair of chromosome segment duplications were identified. SlJMJ07 and SlJMJ08 were located on the same chromosome as the tandem duplication, whereas SlJMJ16 and SlJMJ17 were located on different chromosomes with segmental duplications (Supplementary Figure S3A). The Ka/Ks ratios of SlJMJ07/SlJMJ08 and SlJMJ16/SlJMJ17 were 0.292914 and 0.298160, respectively, indicating that these genes have undergone purifying selection during evolution (Supplementary Figure S3B).

3.2. Gene Structure, Encoded Protein, Conserved Domain, and Cis-Acting Element Analysis of SlJMJ Genes

To better understand the structural diversity of tomato SlJMJ genes, we analysed the gene structure and intron organisation of the 23 SlJMJ members. The genomic sequence lengths of the SlJMJ genes ranged from 1041 bp (SlJMJ06) to 24,132 bp (SlJMJ04) (Supplementary Table S3). Most SlJMJ members contained introns. Specifically, SlJMJ04 contained 32 introns, whereas SlJMJ06 contained no introns (Supplementary Figure S4). The tomato SlJMJ proteins possessed 12 different types of conserved domains: ARIN, F-box, FYRC, FYRN, zf-C5HC2, zf-C2H2, zf-Ring, JmjC, JmjN, PHD, PLU-1, and WRC (Supplementary Figure S5). The KDM3 subfamily members predominantly contained the JmjC, zf-Ring, and WRC domains. KDM4 subfamily members possessed JmjC, JmjN, and zf-C5HC2 domains, whereas the KDM5 subfamily typically contained seven domains: JmjC, JmjN, ARIN, WRC, PHD, FYRN, and FYRC. Notably, JMJD6 subfamily members contained both JmjC and F-box domains.
To further understand their function and regulatory mechanisms, SlJMJ members were subjected to cis-acting element analysis using the PlantCARE database, which revealed 76 different cis-acting elements. Supplementary Table S5 lists the top 22 cis-acting elements. Based on their functions, the top 22 cis-acting elements were divided into four categories: light reaction-related (AE-box, GATA-motif, G-box, TCT-motif, Box4, and GT1-motif), hormone-related (ABRE, ERE, TGA-element, GARE-motif, P-box, CGTCA-motif, TGACG-motif, TCA-element, and ARE), adversity-related (MBS, LTR, O2-site, TC-rich repeats, W-box, and WUN-motif), and growth- and development-related (CAT-box and GCN4-motif). Further statistical analyses revealed that every member of the SlJMJ gene family contained 12–28 cis-elements related to hormonal responses or stress (Figure 1B and Supplementary Table S6). Notably, 22 SlJMJ gene members contained ABRE (a cis-acting element involved in the ABA response), and 20 SlJMJ gene members contained ARE (a cis-acting regulatory element essential for anaerobic induction) (Supplementary Table S6).

3.3. Expression Analysis of Tomato SlJMJ Genes

Based on the Tomato Functional Genomics Database (TFGD), we analysed the expression profiles of SlJMJ genes across four stages of fruit development (0 DPA, 10 DPA, 20 DPA, and 33 DPA), four stages of inflorescence formation (floral buds at 2, 4 and 6 days post floral initiation (2 DBF, 4 DBF and 6 DBF), and floral and inflorescence meristem (FIM)), and six distinct organs (whole root, hypocotyl, cotyledons, young leaves, mature leaves, and vegetative meristems) (Figure 2A and Supplementary Table S1). Most SlJMJ genes exhibited low expression levels in the six organs and during the four stages of fruit development but were highly expressed during the four stages of inflorescence formation, particularly SlJMJ02, SlJMJ04, SlJMJ05, SlJMJ08, SlJMJ17, SlJMJ19, and SlJMJ22. Particularly, SlJMJ17 showed continuous high-level expression during inflorescence formation, which was significantly higher than that during fruit development and in the six different organs.
We also analysed the expression characteristics of SlJMJ genes in tomatoes exposed to tomato spotted wilt virus (TSWV) and abiotic stresses (drought, aluminium, and salt) (Figure 2B) based on TFGD. Under TSWV infection, most SlJMJ genes (12 of 23 genes) did not show differences in expression compared with the controls. However, SlJMJ17 and SlJMJ23 showed moderately elevated expression upon TSWV infection compared to the control. Under salt stress conditions, SlJMJ05 and SlJMJ21 were highly expressed, surpassing their expression levels in control plants. Under drought stress, SlJMJ15 and SlJMJ23 showed higher expression levels than those in the control. Notably, SlJMJ15 exhibited consistently higher expression under drought stress across all three samples (IL2-5, IL9-1, and M82) than in the control. Furthermore, qRT-PCR analyses were conducted on five selected genes (SlJMJ03, SlJMJ08, SlJMJ15, SlJMJ20, SlJMJ21, and SlJMJ23) after PEG and NaCl treatments (Figure 2C and Supplementary Figure S6). Under polyethylene glycol (PEG) treatment, SlJMJ15 exhibited a significant upregulation at the 2 h time point, while the expression levels of other genes showed only minor variations. In contrast, during sodium chloride (NaCl) treatment, the expression of the six genes remained relatively stable. These findings suggest that the expression patterns of these genes under PEG and NaCl treatments are consistent with those reported in previous studies (Figure 2B), thus supporting the reliability of our analysis. Given the marked induction of SlJMJ15 in response to drought stress, further investigation into its functional role in the drought response of tomatoes would be of considerable value.

3.4. Functional Analysis of SlJMJ15 in Tomato Response to Drought Stress

To further analyse the function of SlJMJ15 in the response of tomatoes to drought stress, RNAi and OE transgenic experiments were conducted on the M82 cultivar. Following the expression analysis of transgenic plants, lines exhibiting significant differences in gene expression were selected for comparative evaluation (Figure 3A). Compared with WT plants (leaf length 4.4 cm; leaf width 2.8 cm; plant height 21.1 cm), SlJMJ15-RNAi plants exhibited a severe dwarf phenotype: leaf length 2.2–2.3 cm; leaf width 1.0–1.3 cm; plant height 5.5–5.7 cm. In contrast, SlJMJ15-OE plants (leaf length 5.8–6.0 cm; leaf width 3.1–3.7 cm; plant height 23.7–24.5 cm) only exhibited slight morphological changes (Figure 3B and Supplementary Table S7).
For the drought tolerance analysis, seedlings of similar size and growth status were selected for drought treatment. However, SlJMJ15-RNAi plants exhibited a severe dwarf phenotype, resulting in a significant size difference compared with WT plants. Therefore, only SlJMJ15-OE plants (specifically OE8 and OE10) were selected for further analysis in the subsequent drought treatment experiments. At 3 d post-drought treatment, the SlJMJ15-OE lines exhibited initial signs of wilting, whereas the WT plants remained unaffected and grew normally. At 6 d post-drought treatment, both WT and SlJMJ15-OE lines showed wilting; however, wilting in SlJMJ15-OE lines was more pronounced than that in the WT. At 9 d post-drought treatment, severe wilting was observed in both WT and SlJMJ15-OE lines. Following rehydration, WT plants successfully resumed normal growth, whereas SlJMJ15-OE lines failed to recover and eventually perished (Figure 4A).
Further determination of physiological parameters at 4 d after drought treatment revealed that the REC in the SlJMJ15-OE lines (71.20%) was significantly higher than that in the WT (44.79%). Additionally, the Pro content in the SlJMJ15-OE lines (10.05%) was significantly lower than that in the WT (33.67%) (Figure 4B and Supplementary Table S8). The activities of POD and CAT enzymes in SlJMJ15-OE lines (3367.99 u/g FW; 1228.35 u/g FW) were also significantly lower than those in the WT (4715.25 u/g FW; 2486.65 u/g FW), suggesting that plants overexpressing SlJMJ15 have reduced levels of osmotic stress-related substances and oxidative stress-related enzyme activities associated with drought tolerance. This conclusion was further supported by DAB and NBT staining experiments 4 d after drought treatment (Figure 4C). After DAB and NBT staining, the leaves demonstrated that the number of brown and blue spots in the SlJMJ15-OE lines was significantly greater than that in the WT plants, with darker spots observed in the SlJMJ15-OE lines, indicating that SlJMJ15-OE plants accumulated more reactive oxygen species and experienced greater oxidative damage under drought stress. Collectively, these results demonstrated that SlJMJ15 plays a negative regulatory role in the response of tomato plants to drought stress by modulating Pro levels and antioxidant enzyme activity.

3.5. SlJMJ15 Regulates Expression of Genes Associated with the ABA Pathway to Reduce Tomato Drought Tolerance

ABA is a crucial hormone in plant responses to drought stress, as it enhances drought tolerance by regulating lateral root differentiation and primary root elongation. In the present study, we investigated structural changes in primary root elongation in SlJMJ15-OE lines under ABA treatment (Figure 5). The plant height of SlJMJ15-OE lines (2.86–3.16 cm) did not significantly differ from that of the WT (3.36 cm) after 8 d of cultivation. (Figure 5A,B and Supplementary Table S9). However, the primary root length of SlJMJ15-OE plants (3.85–4.12 cm) was significantly shorter than that of the WT (6.11 cm) after 8 d of cultivation (Figure 5A,C; Supplementary Table S9). However, in the control, there was no significant difference between the SlJMJ15-OE lines and WT in terms of plant height and root length, suggesting that SlJMJ15 OE attenuates primary root elongation under ABA-containing culture conditions.
To further investigate SlJMJ15’s effect on the ABA pathway under drought stress, qRT-PCR analysis was conducted on genes involved in the ABA pathway in the SlJMJ15-OE and WT lines. In plants, NCED3, AO1, and AO2 encode the rate-limiting enzymes of the ABA synthesis pathway [54], whereas PYL4, PYL7, PYL11, PP2C2, ABI3, ABI5, AREB1, and TAS14 are key genes in the ABA signalling pathway [55]. Therefore, we analysed the expression of homologous tomato genes in SlJMJ15-OE and WT plants (Figure 6). In the WT, drought induction significantly upregulated the SlNCED3, SlAO1, SlPYL4, SlPYL7, SlPYL11, SlPP2C2, SlABI3, SlAREB1, and SlTAS14 expressions. SlAO2 was downregulated by drought induction, whereas SlABI5 remained largely unchanged under drought conditions. In SlJMJ15-OE lines, drought induction significantly upregulated SlNCED3 and SlTAS14 but downregulated SlAO1, SlPYL4, SlPYL7, SlPYL11, SlABI5, and SlAREB1. The expression of SlAO2 and SlABI3 did not show significant changes under drought conditions. These results indicated that the SlAO1, SlPYL4, SlPYL7, SlPYL11, and SlAREB1 expression trends were the opposite between SlJMJ15-OE and WT plants. In addition, under drought stress, the SlNCED3 and SlTAS14 expression levels in SlJMJ15-OE lines were significantly higher than those in the WT. The SlAO1, SlPYL4, SlPYL7, SlPYL1, and SlAREB1 expression levels in SlJMJ15-OE lines were significantly lower than those in the WT.

3.6. SlJMJ15 Decreased Drought Tolerance of Tomatoes Through Histone Methylation

SlJMJ15 encodes a demethylase and is most closely related to AtJMJ30 in Arabidopsis (Figure 1A). AtJMJ30 functions as an H3K27me2/me3 demethylase in Arabidopsis. To investigate its role, both SlJMJ15-OE and WT lines were externally treated with 100 μM GSK-J1. In both SlJMJ15-OE lines and the WT, leaf size and plant height in plants treated with GSK-J1 were significantly smaller or lower than those in the control (Figure 7A,B), similar to the morphological changes observed in SlJMJ15-RNAi plants (Figure 3). Additionally, the difference in plant height (D-value) (mean plant height difference between the GSK-J1 treatment and control) was more pronounced in SlJMJ15-OE lines than in the WT (Figure 7B and Supplementary Table S10), suggesting that GSK-J1 inhibits the H3K27me3/me2 demethylation of SlJMJ15. Four days after drought stress treatment, the drought tolerance level in SlJMJ15-OE lines treated with GSK-J1 (3.47–3.73) was higher than that in the control (2–2.07), which was close to that of the WT plants (3.80) treated with GSK-J1 (Figure 7C and Supplementary Table S10). Collectively, these results indicated that tomato plants overexpressing SlJMJ15 exhibited increased susceptibility to drought. Furthermore, H3K27me3/me2 demethylation inhibition mitigated the adverse effects associated with SlJMJ15 OE on the drought stress response.

4. Discussion

As a globally important vegetable crop, tomatoes play a pivotal role in ensuring food security and supporting the agricultural economy. However, drought stress induced by climate change severely threatens its production and quality, potentially resulting in a 50% reduction in yield [29]. This underscores the pressing need for research on the drought tolerance mechanisms in tomatoes. Epigenetic regulation is a key process in plant stress responses, with histone demethylases being particularly important for the molecular resistance mechanisms that regulate the expression of stress-responsive genes. The JMJ gene family, which encodes proteins containing the JmjC domain, is critical for maintaining the dynamic balance of histone methylation. This gene family is widely distributed across animal and plant genomes and plays an essential role in epigenetic regulation. Currently, genome-wide identification and characterisation of JMJ gene families have been completed in a few species, including Arabidopsis [18], rice [56], and maize [20]. However, SlJMJ genes in tomatoes have not yet been systematically identified or characterised with respect to their roles in the drought stress response.
In the present study, 23 SlJMJ genes were identified in the tomato genome database. We found that the number of JMJ family members was significantly different among species: 48 members in soybean [57], 24 members in wheat [58], 22 members in strawberry [59], 21 members in grape [60], and 16 members in banana [61]. Phylogenetic analysis showed that the SlJMJ gene family can be classified into five subfamilies: KDM3, KDM4, KDM5, JMJD6, and JmjC domains. This finding was consistent with that of the AtJMJ family in Arabidopsis. However, in Rosa chinensis [19] and Brassica napus [62], the KDM5 subfamily is divided into KDM5A and KDM5B, whereas the JmjC-only subfamily is divided into JmjC domain-only A and B. In addition, among the tomato SlJMJ subfamilies, KDM3 was the largest, with eight members, which is consistent with the findings in upland cotton [63], grape [60], and birch [64].
Cis-acting elements in the promoter regions play roles in gene transcription and expression [65]. ABRE and ARE cis-acting elements are involved in plant stress responses [66]. There are 22 SlJMJ gene members that contain ABRE (involved in the ABA response) and 20 SlJMJ gene members that contain ARE (essential for anaerobic induction). Notably, SlJMJ15 was enriched in stress-responsive cis-elements, with nine ABRE and three ARE motifs (Supplementary Table S6). These 12 stress-associated cis-acting elements represent the highest density among the 23 SlJMJ members. Moreover, SlJMJ15 expression was specifically induced under salt and drought stress conditions. The closely related homologous gene AtJMJ30 reportedly plays a role in balancing the ABA and BR signalling pathways in response to stress [67], suggesting that SlJMJ15 may also be involved in salt and drought responses in tomatoes.
Drought is a major abiotic stress that tomato plants must cope with during their growth and development. qRT-PCR assays showed that the SlJMJ15 expression level was significantly upregulated by PEG treatment. SlJMJ15-RNAi plants exhibited a severe dwarf phenotype, which is an adaptive trait in plants, enabling them to conserve energy under adverse conditions [68,69]. SlJMJ15-OE plants displayed a more severe wilting phenotype than the WT plants after drought stress treatment, suggesting that SlJMJ15 plays a negative regulatory role in the response of tomato plants to drought stress. However, SlJMJ15 OE improved Cd tolerance through the GSH-PC pathway in Arabidopsis [28], suggesting that the effects of SlJMJ15 on plant responses differ across stress types.
ABA is a crucial hormone involved in plant responses to adverse stress. Under ABA-containing culture conditions, the primary root length of SlJMJ15-OE plants was shorter than that of WT plants, indicating that SlJMJ15 may be involved in drought response via the ABA pathway. PYL, a receptor for ABA, plays an important role in the ABA pathway [55]. qRT-PCR analysis revealed that SlPYL4, SlPYL7, and SlPYL11 expression was lower in SlJMJ15-OE plants than in WT plants under drought conditions. In Arabidopsis, AtPYL9 promotes drought resistance and leaf senescence [70]. In tomato, SlPYL9-OE lines showed increased drought tolerance [71]. AREB1 is a basic leucine zipper transcription factor that binds to the ABA element (ABRE) motif in the promoter region of ABA-inducible genes [72]. AtAREB1 OE can improve soybean responses to water deficit [73]. In the present study, SlAREB1 expression was downregulated in SlJMJ15-OE plants under drought stress. The dehydrin gene SlTAS14 alleviates osmotic stress caused by drought and salinity in tomatoes [74]. In our study, SlTAS14 expression was upregulated in SlJMJ15-OE plants, suggesting that SlJMJ15 plays a role in the response of tomato plants to drought stress via the ABA signalling pathway.
As SlJMJ15 is a member of the SlJMJ gene family encoding histone demethylases in tomato, and its closely related protein AtJMJ30 has been reported to remove H3K27me3/me2 from BZR1 [67], GSK-J1 treatment was conducted in the present study. In the SlJMJ15-OE lines, the drought tolerance level of plants treated with GSK-J1 was higher than that of the control group, which was close to that of the WT plants (Figure 7C). In tomatoes, SlJMJ6 promotes fruit ripening by removing H3K27 methylation of ripening-related genes [26], whereas SlJMJ7 regulates tomato fruit ripening via crosstalk between H3K4me3 and DML2-mediated DNA demethylation [27]. In Arabidopsis, AtJMJ27-mediated histone H3K9 demethylation positively regulates drought-stress responses [75]. Therefore, we hypothesised that GSK-J1 inhibits the demethylating activity of SlJMJ15, thereby alleviating the adverse effects associated with its OE.
Under drought stress, histone demethylation and DNA demethylation may act synergistically on drought-responsive gene clusters, efficiently regulating gene expression through chromatin structural remodelling and exposure of transcription factor binding sites [76,77]. For instance, the JmjC domain-containing protein JMJ25/IBM1 prevents silencing of moderately transcribed and constitutively expressed genes by reducing H3K9 dimethylation and non-CG DNA methylation levels [78]. Therefore, our study suggests that SlJMJ15 may contribute to reduced drought tolerance in tomatoes via its demethylation function, which affects the expression of genes associated with the ABA signalling pathway. In the future, we will further analyse changes in the methylation levels of related genes to identify the target genes of SlJMJ15. Overall, this study provides insights into the roles of SlJMJ family genes in tomato plant growth and abiotic stress responses, which can facilitate further investigation of their underlying molecular mechanisms and contribute to improving stress tolerance in tomatoes.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11101148/s1, Supplementary Figure S1. Phylogenetic relationships of the SlJMJ genes. Supplementary Figure S2. The chromosome distribution of the identified SlJMJ genes in tomato. Supplementary Figure S3. Covariance analysis of the tomato SlJMJ genes. Supplementary Figure S4. Gene structure analysis of the tomato SlJMJ genes. Supplementary Figure S5. Domain composition of the SlJMJ proteins in tomato. Supplementary Figure S6. The qRT-PCR assays of SlJMJ03, SlJMJ08, SlJMJ15, SlJMJ20, SlJMJ21 and SlJMJ23 in NaCl treatment (in tomato M82); Supplementary Tables S1–S10: Supplementary Table S1. The expression analysis based on RNA-seq data of SlJMJs in fruit development, inflorescence formation, different organs, and stress types. Supplementary Table S2-1. The primers used in this study. Supplementary Table S2-2. The qRT-PCR assays of SlJMJ03, SlJMJ08, SlJMJ15, SlJMJ20, SlJMJ21 and SlJMJ23 in PEG and NaCl treatment (in tomato M82). Supplementary Table S2-3. Expression analysis of ABA related genes in SlJMJ15-overexpression (OE) plants and wiled type (WT) before and after drought treatment. Supplementary Table S3. The basic information of the JMJ gene family in tomato. Supplementary Table S4. Statistical analysis of members of the JMJ subfamily in Arabidopsis, tomato, and rice. Supplementary Table S5. Functionally annotated of cis-elements identified in the promoters of SlJMJ genes. Supplementary Table S6. Statistical analysis of cis-elements related to hormonal response or stress in different SlJMJ genes. Supplementary Table S7-1. The morphological changes assays of SlJMJ15-RNAi and SlJMJ15-OE tomato plants. Supplementary Table S7-2. The SlJMJ15 expression level of the wild type (WT), SlJMJ15-RNAi and SlJMJ15-overexpression (OE) materials. Supplementary Table S8. Determination of physiological parameters in SlJMJ15-OE lines and WT after drought treatment. Supplementary Table S9. Root length and plant height of the SlJMJ15-OE tomato lines after exogenous ABA treatment. Supplementary Table S10. Plant height and drought tolerance level of the SlJMJ15-OE tomato lines and WT after exogenous GSK-J1 treatment.

Author Contributions

L.W. contributed to the experiments and writing of the manuscript. H.Z. contributed to the GSK-J1 treatment experiments. Q.Y. contributed to the over-expression transgenic experiment in M82. F.L. contributed to the identification and bioinformatics analysis of the tomato JMJ gene family. Y.L. contributed to the qPCR of genes associated with the ABA pathway. J.X. contributed to the construction of the Plasmids used in the study. D.X. contributed to RNA isolation and Real-Time Quantitative PCR assays. Y.P. and X.Z. revised the manuscript. J.L. supervised the entire process. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by ‘Fundamental Research Funds for the Central Universities’ (SWU-KQ22041), Special Key Project of Technological Innovation and Application Development of Chongqing (CSTB2023TIAD-KPX0026), and the College Students’ Innovative Entrepreneurial Training Plan Program (S202410635351).

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

The following abbreviations are used in this manuscript:
α-KGα-ketoglutaric acid
ABAabscisic acid
AlAl-treated
CATcatalase
CKblank control
COTYLcotyledon
DAB3,3′-diaminobenzidine
DSdrought stress
HYPOhypocotyl
JmjCjumonji C
LSD1lysine-specific demethylase 1
MERIvegetative meristem
MLmature leaves
MSMurashige–Skoog
OEoverexpression
NBTnitrotetrazolium blue chloride
PODperoxidase
Proproline
RECrelative electrical conductivity
ROOTwhole root
RNAiRNA interference
RT-qPCRreverse transcription-quantitative PCR
STsalt-treated
TSWVtomato spotted wilt virus
WTWild type
YLyoung leaves

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Figure 1. Phylogenetic analysis and cis-acting elements of identified SlJMJ genes. (A) Phylogenetic analysis was performed using MEGA 7.0. Different groups are shown in differently coloured branch lines and arcs: brown, KDM3; blue, KDM4; red, KDM5; yellow, JMJD6; green, JMJC. The circles, stars, and squares represent SlJMJs, OsJMJs, and AtJMJs in order. (B) Main cis-acting elements of SlJMJ genes. The promoter region extends 1500 bp upstream of the transcriptional start site for the cis-acting regulatory analysis of gene promoters. Different coloured rectangles at different positions in the gene promoter region represent cis-acting elements.
Figure 1. Phylogenetic analysis and cis-acting elements of identified SlJMJ genes. (A) Phylogenetic analysis was performed using MEGA 7.0. Different groups are shown in differently coloured branch lines and arcs: brown, KDM3; blue, KDM4; red, KDM5; yellow, JMJD6; green, JMJC. The circles, stars, and squares represent SlJMJs, OsJMJs, and AtJMJs in order. (B) Main cis-acting elements of SlJMJ genes. The promoter region extends 1500 bp upstream of the transcriptional start site for the cis-acting regulatory analysis of gene promoters. Different coloured rectangles at different positions in the gene promoter region represent cis-acting elements.
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Figure 2. Expression-specific analysis of tomato SlJMJ genes. (A) Heat map of SlJMJ expression during fruit development, inflorescence formation, and in different organs. 0 DPA, 0 d post-anthesis fruit or anthesis flowers; 10 DPA, 10 d post-anthesis fruit; 20 DPA, 20 d post-anthesis fruit; 33 DPA, 33 d post-anthesis fruit; COTYL, cotyledons; HYPO, hypocotyl; MERI, vegetative meristems; ML, mature leaves; ROOT, whole root; YL, young leaves; 2 DBF, floral buds at 2 d post floral initiation; 4 DBF, floral buds at 4 days post floral initiation; 6 DBF, floral buds at 6 d post floral initiation; FIM, floral and inflorescence meristem. The colour scale represents FPKM-normalised Log2-transformed counts. (B) Heat map of SlJMJ expression under different stress conditions. CK, blank control; DS, drought stress; Al, Al-treated; ST, salt-treated; TSWV-R, root after TSWV infection; TSWV-L, leaf after TSWV infection. Colour scale represents FPKM-normalised Log2-transformed counts. (C) qRT-PCR assays for SlJMJ03, SlJMJ08, SlJMJ15, SlJMJ20, SlJMJ21, and SlJMJ23 after PEG treatment (in tomato M82). The data are presented as mean ± standard deviation (n = 3). Letters indicate significant differences according to one-way ANOVA (Tukey’s test; p < 0.05).
Figure 2. Expression-specific analysis of tomato SlJMJ genes. (A) Heat map of SlJMJ expression during fruit development, inflorescence formation, and in different organs. 0 DPA, 0 d post-anthesis fruit or anthesis flowers; 10 DPA, 10 d post-anthesis fruit; 20 DPA, 20 d post-anthesis fruit; 33 DPA, 33 d post-anthesis fruit; COTYL, cotyledons; HYPO, hypocotyl; MERI, vegetative meristems; ML, mature leaves; ROOT, whole root; YL, young leaves; 2 DBF, floral buds at 2 d post floral initiation; 4 DBF, floral buds at 4 days post floral initiation; 6 DBF, floral buds at 6 d post floral initiation; FIM, floral and inflorescence meristem. The colour scale represents FPKM-normalised Log2-transformed counts. (B) Heat map of SlJMJ expression under different stress conditions. CK, blank control; DS, drought stress; Al, Al-treated; ST, salt-treated; TSWV-R, root after TSWV infection; TSWV-L, leaf after TSWV infection. Colour scale represents FPKM-normalised Log2-transformed counts. (C) qRT-PCR assays for SlJMJ03, SlJMJ08, SlJMJ15, SlJMJ20, SlJMJ21, and SlJMJ23 after PEG treatment (in tomato M82). The data are presented as mean ± standard deviation (n = 3). Letters indicate significant differences according to one-way ANOVA (Tukey’s test; p < 0.05).
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Figure 3. Morphological changes in SlJMJ15-RNAi and SlJMJ15-overexpression (OE) tomato plants. (A) Phenotype and SlJMJ15 expression levels of the WT, SlJMJ15-RNAi, and SlJMJ15-OE materials. Scale bar = 5 cm. For SlJMJ15 expression analysis, three biological replicates were investigated for each sample, and the mean was calculated. (B) Leaf length, leaf width, and plant height of WT, SlJMJ15-RNAi, and SlJMJ15-OE plants. Twenty plants were investigated for each sample, and the mean was calculated. Asterisks indicate the significance level based on Tukey’s multiple comparison test (** 0.005 ≤ p < 0.01, *** 0.001 ≤ p < 0.005, **** p ≤ 0.001).
Figure 3. Morphological changes in SlJMJ15-RNAi and SlJMJ15-overexpression (OE) tomato plants. (A) Phenotype and SlJMJ15 expression levels of the WT, SlJMJ15-RNAi, and SlJMJ15-OE materials. Scale bar = 5 cm. For SlJMJ15 expression analysis, three biological replicates were investigated for each sample, and the mean was calculated. (B) Leaf length, leaf width, and plant height of WT, SlJMJ15-RNAi, and SlJMJ15-OE plants. Twenty plants were investigated for each sample, and the mean was calculated. Asterisks indicate the significance level based on Tukey’s multiple comparison test (** 0.005 ≤ p < 0.01, *** 0.001 ≤ p < 0.005, **** p ≤ 0.001).
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Figure 4. Drought tolerance analysis of SlJMJ15-overexpression (OE) tomato lines. (A) Phenotypic analysis of the SlJMJ15-OE lines (OE8 and OE10) under drought stress. 1 d, on the first day post-drought treatment; 3 d, on the third day post-drought treatment; 6 d, on the sixth day post-drought treatment; 9 d, on the ninth day post-drought treatment; R3 d, on the third day after re-watering. Scale bar = 6 cm. (B) Physiology of SlJMJ15-OE lines (OE8 and OE10) 4 d after drought treatment. REC, relative electrical conductivity; POD, peroxidase enzyme activity; CAT, catalase enzyme activity; Pro, proline (Pro) content. Three biological replicates were investigated for each sample, and the mean was calculated. Asterisks indicate the significance level based on Tukey’s multiple comparison test (* 0.01 ≤ p < 0.05, ** 0.005 ≤ p < 0.01, **** p ≤ 0.001). (C) DAB and NBT staining of leaves in SlJMJ15-OE lines and WT plants 4 d post-drought treatment. Control, leaves stained without drought stress treatments; DAB, leaves stained using DAB; NBT, leaves stained using NBT. Scale bar = 1 cm.
Figure 4. Drought tolerance analysis of SlJMJ15-overexpression (OE) tomato lines. (A) Phenotypic analysis of the SlJMJ15-OE lines (OE8 and OE10) under drought stress. 1 d, on the first day post-drought treatment; 3 d, on the third day post-drought treatment; 6 d, on the sixth day post-drought treatment; 9 d, on the ninth day post-drought treatment; R3 d, on the third day after re-watering. Scale bar = 6 cm. (B) Physiology of SlJMJ15-OE lines (OE8 and OE10) 4 d after drought treatment. REC, relative electrical conductivity; POD, peroxidase enzyme activity; CAT, catalase enzyme activity; Pro, proline (Pro) content. Three biological replicates were investigated for each sample, and the mean was calculated. Asterisks indicate the significance level based on Tukey’s multiple comparison test (* 0.01 ≤ p < 0.05, ** 0.005 ≤ p < 0.01, **** p ≤ 0.001). (C) DAB and NBT staining of leaves in SlJMJ15-OE lines and WT plants 4 d post-drought treatment. Control, leaves stained without drought stress treatments; DAB, leaves stained using DAB; NBT, leaves stained using NBT. Scale bar = 1 cm.
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Figure 5. Effect of exogenous ABA treatment on SlJMJ15-overexpression (OE) and wild-type (WT) tomato plants. (A) Phenotypic analysis of SlJMJ15-OE lines cultured in the presence of ABA. Control, MS medium without 250 μM ABA (2 μMol/L); 2 μMol/L ABA, MS medium supplemented with 250 μM ABA (2 μMol/L). Scale bar = 5 cm. (B) Plant height analysis of SlJMJ15-OE lines cultured in the presence of ABA. The values were measured after 8 d of cultivation in MS medium. (C) Primary root length of SlJMJ15-OE lines cultured in the presence of ABA. The values were measured after 8 d of cultivation in MS medium. All root length and plant height data represent the averages of three biological and three technical replicates. Asterisks indicate the significance level based on Tukey’s multiple comparison test (* 0.01 ≤ p < 0.05).
Figure 5. Effect of exogenous ABA treatment on SlJMJ15-overexpression (OE) and wild-type (WT) tomato plants. (A) Phenotypic analysis of SlJMJ15-OE lines cultured in the presence of ABA. Control, MS medium without 250 μM ABA (2 μMol/L); 2 μMol/L ABA, MS medium supplemented with 250 μM ABA (2 μMol/L). Scale bar = 5 cm. (B) Plant height analysis of SlJMJ15-OE lines cultured in the presence of ABA. The values were measured after 8 d of cultivation in MS medium. (C) Primary root length of SlJMJ15-OE lines cultured in the presence of ABA. The values were measured after 8 d of cultivation in MS medium. All root length and plant height data represent the averages of three biological and three technical replicates. Asterisks indicate the significance level based on Tukey’s multiple comparison test (* 0.01 ≤ p < 0.05).
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Figure 6. Expression change analysis of ABA-related genes in SlJMJ15-overexpression (OE) and wild-type (WT) plants before and after drought treatment. At 4 d post-drought treatment, total RNA was extracted using the RNeasy Kit (Qiagen, Inc., Hilden, Germany). All RT-qPCR data represent the average of three biological replicates and three technical replicates. Asterisks indicate the significance level based on Tukey’s multiple comparison test (* 0.01 ≤ p < 0.05, ** 0.005 ≤ p < 0.01, *** 0.001 ≤ p < 0.005, **** p ≤ 0.001).
Figure 6. Expression change analysis of ABA-related genes in SlJMJ15-overexpression (OE) and wild-type (WT) plants before and after drought treatment. At 4 d post-drought treatment, total RNA was extracted using the RNeasy Kit (Qiagen, Inc., Hilden, Germany). All RT-qPCR data represent the average of three biological replicates and three technical replicates. Asterisks indicate the significance level based on Tukey’s multiple comparison test (* 0.01 ≤ p < 0.05, ** 0.005 ≤ p < 0.01, *** 0.001 ≤ p < 0.005, **** p ≤ 0.001).
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Figure 7. Effect of exogenous demethylation inhibitor (GSK-J1) treatment on the SlJMJ15-overexpression (OE) tomato lines and wild type (WT). (A) Phenotypes of WT, OE10, and OE08 plants before and after drought treatment. Control, blank control treated with water; GSK-J1, experimental treatment with 100 μM GSK-J1 methylating inhibitors. Scale bar = 5 cm. (B) Plant height of SlJMJ15-OE tomato lines and WT plants after exogenous GSK-J1 treatment. At 4 d after soil drought treatment, the height of 15 plants in each sample was measured. (C) Drought tolerance levels of SlJMJ15-OE tomato lines and WT plants after exogenous GSK-J1 treatment. The drought tolerance level reflects the leaf growth status of plants 4 d after drought treatment and is categorised as follows: 1, more than three completely dead leaves; 2, two completely dead leaves; 3, one completely dead leaf with yellowing and wilting; 4, no completely dead leaves, with only a small area showing yellowing and wilting. At 4 d after soil drought treatment, the drought tolerance level of 15 plants per sample was assessed. Asterisks indicate the significance level based on Tukey’s multiple comparison test (* 0.01 ≤ p < 0.05, ** 0.005 ≤ p < 0.01, *** 0.001 ≤ p < 0.005).
Figure 7. Effect of exogenous demethylation inhibitor (GSK-J1) treatment on the SlJMJ15-overexpression (OE) tomato lines and wild type (WT). (A) Phenotypes of WT, OE10, and OE08 plants before and after drought treatment. Control, blank control treated with water; GSK-J1, experimental treatment with 100 μM GSK-J1 methylating inhibitors. Scale bar = 5 cm. (B) Plant height of SlJMJ15-OE tomato lines and WT plants after exogenous GSK-J1 treatment. At 4 d after soil drought treatment, the height of 15 plants in each sample was measured. (C) Drought tolerance levels of SlJMJ15-OE tomato lines and WT plants after exogenous GSK-J1 treatment. The drought tolerance level reflects the leaf growth status of plants 4 d after drought treatment and is categorised as follows: 1, more than three completely dead leaves; 2, two completely dead leaves; 3, one completely dead leaf with yellowing and wilting; 4, no completely dead leaves, with only a small area showing yellowing and wilting. At 4 d after soil drought treatment, the drought tolerance level of 15 plants per sample was assessed. Asterisks indicate the significance level based on Tukey’s multiple comparison test (* 0.01 ≤ p < 0.05, ** 0.005 ≤ p < 0.01, *** 0.001 ≤ p < 0.005).
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Wu, L.; Zhao, H.; Xu, J.; Lin, F.; Yan, Q.; Liang, Y.; Xu, D.; Pan, Y.; Zhang, X.; Li, J. The SlJMJ15, a Putative Histone Demethylase Gene, Acts as a Negative Regulator of Drought Tolerance in Tomato. Horticulturae 2025, 11, 1148. https://doi.org/10.3390/horticulturae11101148

AMA Style

Wu L, Zhao H, Xu J, Lin F, Yan Q, Liang Y, Xu D, Pan Y, Zhang X, Li J. The SlJMJ15, a Putative Histone Demethylase Gene, Acts as a Negative Regulator of Drought Tolerance in Tomato. Horticulturae. 2025; 11(10):1148. https://doi.org/10.3390/horticulturae11101148

Chicago/Turabian Style

Wu, Lang, Hanling Zhao, Jiajia Xu, Fasen Lin, Qingxia Yan, Yan Liang, Danyang Xu, Yu Pan, Xingguo Zhang, and Jinhua Li. 2025. "The SlJMJ15, a Putative Histone Demethylase Gene, Acts as a Negative Regulator of Drought Tolerance in Tomato" Horticulturae 11, no. 10: 1148. https://doi.org/10.3390/horticulturae11101148

APA Style

Wu, L., Zhao, H., Xu, J., Lin, F., Yan, Q., Liang, Y., Xu, D., Pan, Y., Zhang, X., & Li, J. (2025). The SlJMJ15, a Putative Histone Demethylase Gene, Acts as a Negative Regulator of Drought Tolerance in Tomato. Horticulturae, 11(10), 1148. https://doi.org/10.3390/horticulturae11101148

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