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Article

Sly-miR398 Participates in Heat Stress Tolerance in Tomato by Modulating ROS Accumulation and HSP Response

by
Baoyu Li
1,
Peiwen Wang
1,
Shuaijing Zhao
1,
Jiaqi Dong
1,
Shengming Mao
1,
Xuyongjie Zhu
1,
Tiantian Yuan
1,
Haiying Qiu
1,
Long Cao
1,
Yunmin Xu
1,2,3,
Yong He
1,2,3,
Zhujun Zhu
1,2,3 and
Guochao Yan
1,2,3,*
1
College of Horticulture Science, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China
2
Key Laboratory of Quality and Safety Control for Subtropical Fruit and Vegetable, Ministry of Agriculture and Rural Affairs, Hangzhou 311300, China
3
Collaborative Innovation Center for Efficient and Green Production of Agriculture in Mountainous Areas of Zhejiang Province, Hangzhou 311300, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(2), 294; https://doi.org/10.3390/agronomy15020294
Submission received: 19 December 2024 / Revised: 21 January 2025 / Accepted: 21 January 2025 / Published: 24 January 2025
(This article belongs to the Section Horticultural and Floricultural Crops)
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Abstract

:
Heat stress is one of the most important environmental problems in agriculture, which severely restricts the growth and yield of plants. In plants, microRNA398 (miR398) negatively regulates the activity of superoxide dismutase (SOD) by modulating the expression of its coding genes (CSDs) post-transcriptionally, thereby regulating reactive oxygen species (ROS) homeostasis and stress resistance. In this study, the role of miR398 in heat stress tolerance in tomato was investigated. Under heat stress, the expression of miR398 was upregulated in tomato, while the expression of its target genes (CSD1 and CSD2) and SOD activity was downregulated. Furthermore, by comparing the heat stress response in wild type (WT) and a transgenic line overexpressing MIR398 (miR398-OE), the results showed that overexpression of miR398 promoted tomato growth and the expression of genes encoding heat shock factor (HSF, transcription factor) and heat shock protein (HSP) under heat stress. Meanwhile, downregulated activity of antioxidant enzymes, including SOD, catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX), and enhanced ROS accumulation was observed in miR398-OE compared with that in WT under heat stress. Further study using dimethylthiourea (DMTU, a ROS scavenger) indicated that the enhanced plant growth and expression of HSFs/HSPs was based on the promoted accumulation of ROS in miR398-OE. Overall, the results of this study revealed that the upregulated expression of miR398 in response to heat stress would modulate the antioxidant system and enhance ROS accumulation, thereby enhancing the expression of HSFs and HSPs and heat stress tolerance in tomato.

1. Introduction

Heat stress is one of the major environmental stresses in agricultural practice [1]. In plants grown under high ambient temperatures, heat stress disrupts protein functional conformation and results in metabolic disorder, thereby limiting plant growth and productivity [2,3,4]. Particularly, it has been estimated that the global average temperature will increase by approximately 0.3 °C every decade, and the constantly rising environmental temperature induces severe yield loss and threatens food security worldwide [5,6,7].
As sessile organisms, plants have evolved multiple mechanisms in heat stress acclimation at morphological, physiological, and molecular levels [8,9,10]. It has been demonstrated that heat shock proteins (HSPs) play an important role in the thermotolerance of plants. Under heat stress, HSPs would be activated via multiple mechanisms such as heat shock transcription factors (HSFs) and reactive oxygen species (ROS) and act as molecular chaperones, protecting proteins from heat stress-induced denaturation and enhancing plant antioxidant system activity [11,12]. In general, HSFs exist in the form of HSF-HSP complex, which would be released by heat stress-induced denatured proteins through drawing HSP away, thereby resulting in the promotion of HSP transcripts in heat stress response. Besides, plants would enhance ROS generation and successively activate the formation of HSF trimers and enhance HSP expression [13,14]. In conclusion, the ROS-HSF-HSP feedback loop is a key mechanism in plant heat stress resistance.
MicroRNAs (miRNAs) are ~21-nucleotide RNAs, which are derived from the stem-loop regions of long primary transcripts and generally direct the cleavage of their complementary mRNAs, thereby regulating the expression of target genes at the post-transcriptional level [15,16]. In the past several decades, after the first identification of miRNAs in plants, the pivotal roles of miRNAs in plant growth, development, regeneration, nutrition homeostasis, and stress acclimation have been extensively recognized [15,17]. As an important member of miRNAs in plants, microRNA398 (miR398), which was first identified in Arabidopsis [18], negatively modulates the expression of CSDs (encoding copper-zinc superoxide dismutase, Cu-Zn SOD), CCS (encoding copper chaperone for CSDs) and COX5b-1 (encoding chlorophyll C oxidase subunit) genes, thereby participating the regulation of SOD activity [19]. As the first line of plant antioxidant defense system, SOD converts superoxide anion (O2·−) into hydrogen peroxide (H2O2) and participates in the sophisticated regulation of ROS balance in plants [20,21]. Due to the multiple functions of ROS in plants, it is not surprising that miR398 participates in the regulation of plant growth, development, and stress resistance including heat stress [22,23].
However, it is worth noting that the expression pattern of miR398 in response to stresses and its successive regulation on CSDs differ among stress types. Particularly, in the context of heat stress, miR398 is shown to be involved in HSP response and thermotolerance in Arabidopsis [24,25] and Chinese cabbage [26], while the response of miR398 to heat stress also differs with plant species. Under heat stress, the expression of miR398 is upregulated in Arabidopsis while downregulated in Chinese cabbage [26]. Therefore, the role of miR398 in the ROS-HSF-HSP and heat stress resistance in different plant species still needs further investigation. Besides, it is important to note that the cooperation of various antioxidant enzymes plays a critical role in scavenging and maintaining ROS, and the members of the antioxidant system may interact with one another in a complex antioxidant system [27,28]. Therefore, the regulatory effect of miR398 on SOD could sequentially affect the activity of other antioxidant enzymes under heat stress, thereby modulating ROS balance and participating in the ROS-HSF-HSP feedback loop.
Tomato (Solanum lycopersicum L.) is an important horticultural crop worldwide, which suffers from heat stress severely. In agricultural practice, the optimum temperature regime for tomato growth is 25–28 °C and 16–18 °C during the day and the night, respectively, while a daily average temperature above 32 °C would limit tomato growth and productivity [29,30]. In the present study, we evaluated the response of miR398 to heat stress in tomato, and examined the role of miR398 in heat stress tolerance with MIR398 (miR398-OE) in transgenic plants. The results indicate that the expression of miR398 was upregulated in tomato under heat stress, thereby downregulating antioxidant enzyme activity and facilitating ROS accumulation, which would subsequently enhance the HSF/HSP response and heat stress tolerance in tomato.

2. Materials and Methods

2.1. Plant Materials and Growth Condition

Tomato (Solanum lycopersicum L.) seeds of wild type (WT, ‘Microtom’) and transgene overexpressing MIR398 (miR398-OE) were derived from our earlier work [31]. After sterilization with 10% (v/v) H2O2 solution and washed thoroughly with deionized water 3 times, tomato seeds were placed on moist filter paper for 2 d of germination in the dark at 28 °C. Then, tomato seeds were sown on a growth substrate (Golden No. 3, Jinhai, China) and grown in an environmental-controlled chamber, while the growth conditions were set as follows: 70% relative humidity, 16/8 h, and 28/20 °C for day/night period. After 4 weeks of growth, tomato seedlings of WT and miR398-OE were treated with control (CK, 28/20 °C for day/night period) or heat stress (HS, 42/38 °C for day/night period) for 2 d.

2.2. Biomass, Relative Water Content, and MDA Content

After 2 d of treatment, tomato seedlings were harvested, and dry weight (DW) was measured after oven-dry at 120 °C for 1 h and at 80 °C for 72 h. Relative water content (RWC) was measured as described by Weatherley [32]. Briefly, the fresh weight (FW), turgid weight (TW), and dry weight (DW) of samples were measured, and RWC was calculated as (FW − DW)/(TW − DW). As for relative electrolyte leakage (REL), the electrical conductivity (EC) of samples was determined after incubation in water on a shaker at 30 °C for 6 h (EC1) and further incubation in boiling water for 30 min (EC2) using an electrical conductivity meter (FE38, Mettler Toledo, Switzerland). REL was calculated as EC1/EC2 [33]. In addition, the content of malonaldehyde (MDA) was measured using the trichloroacetic acid-thiobarbituric acid (TCA-TBA) method with an UV spectrophotometer (UV2600, Shimadzu, Kyoto, Japan) [34].

2.3. ROS Accumulation

To evaluate the accumulation of ROS in WT and miR398-OE under heat stress, nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) staining was conducted in this study for the visualization of superoxide anions (O2·−) and hydrogen peroxide (H2O2) as described by Jabs et al. [35] and Vanacker et al. [36], respectively. Moreover, the content of O2·− and H2O2 was quantitatively determined using the nicotinamide adenine dinucleotide-phenazine methosulfate (NADH-PMS) method [37] and hydroxylamine oxidation method [38], respectively.

2.4. Antioxidant Enzyme Activity

Fresh samples were homogenized with phosphate buffer solution (50 mM, pH 7.8, containing 0.2 mM ethylenediamine tetraacetic acid and 2% (w/v) polyvinyl pyrrolidone). Then, the supernatant was collected after centrifugation at 12,000 rpm for 10 min at 4 °C [39]. Superoxide dismutase (SOD) activity was determined by measuring the photochemical reduction of NBT at 560 nm, and SOD activity per unit (U) was defined as 50% inhibition of NBT reduction [40]. As for catalase (CAT) activity, the raw extract was mixed with phosphate butter solution (25 mM, pH 7.0, containing 10 mM H2O2), and the decrement of H2O2 was measured at 240 nm [41]. The activity of ascorbate peroxidase (APX) was measured by the ascorbate oxidation method according to Nakano and Asada [42]. The activity of guaiacol peroxidase (GPOD) was measured using the method described by Egley et al. [43].

2.5. H2O2 Scavenger Treatment

To evaluate the role of H2O2 in miR398-mediated heat stress response, tomato seedlings (4 weeks old) of WT and miR398-OE were pretreated with dimethylthiourea (DMTU, H2O2 scavenger, 10 mM, CAS: 534-13-4, Leyan, China) in the form of foliar spray 2 d before heat stress treatment for 2 times (one time each d). Then, the expression of HSFs and HSPs in WT and miR398-OE were measured after 3 h of heat stress treatment, and tomato growth parameters, including DW, RWC, and MDA, content were measured after 2 d of heat stress treatment.

2.6. Gene Expression

Frozen tomato samples were used for total RNA extraction using Trizol reagent (Invitrogen, Thermo Fisher, Waltham, MA, USA). The miRNA and RNA were transformed into cDNA after verification of purity and integrity by using the first strand cDNA synthesis kit (Vazyme, Nanjing, China) and HiScriptIII 1st strand cDNA synthesis kit (Vazyme, China). A quantitative PCR system (qTower3, Analytik jena, Jena, Germany) was used to conduct quantitative real-time PCR with chamQ SYBR qPCR master mixture (Vazyme, China). Using ΔΔCt method, the relative expression rate was calculated. U6 was selected as the internal reference genes for miR398, and Actin for CSD1, CSD2, HSFA1a, HSFA2, HSP17.6, HSP70-2, HSP90-1, CAT, APX, and GPOD. The sequences of primers are listed in Table S1.

2.7. Statistical Analysis

All data were computed with Excel 2019 (Microsoft, USA) and analysis of variance (ANOVA) analysis. The mean values were compared with the least significant difference (LSD) method, and the difference was statistically significant at p < 0.05.

3. Results

3.1. Response of miR398 to Heat Stress in Tomato

To investigate the response of miR398 to heat stress in tomato, the expression of miR398, CSD1, CSD2, and the activity of SOD were measured at 1 and 3 h after treatment (HAT). In general, the expression of miR398 was significantly upregulated in tomato under heat stress at 1 and 3 HAT, while the upregulation was more significant at 1 HAT (Figure 1A). Along with the upregulated expression of miR398, the expression of CSD1 and CSD2 was downregulated at 1 and 3 HAT (Figure 1B,C). In addition, the activity of SOD was downregulated at 3 HAT (although not at 1 HAT) (Figure 1D). Overall, these results showed that miR398 was upregulated in response to heat stress and subsequently downregulated the expression of CSD1/CSD2 and the activity of SOD in tomato (Figure 1).

3.2. miR398-OE Was More Tolerant to Heat Stress than WT

Based on the results mentioned above, a transgenic line overexpressing MIR398 (miR398-OE) was used in this study. The growth parameters, including shoot dry weight (DW), relative water content (RWC), relative electrolyte leakage (REL), and malonaldehyde (MDA) content under heat stress, were comparatively evaluated between wild type (WT) and miR398-OE. Under control treatment (CK), no significant difference in seedling growth parameters between WT and miR398-OE was observed. After 2 d of heat stress treatment, the growth of both WT and miR398-OE was significantly suppressed, while heat stress induced more significant growth inhibition and decline of shoot DW in WT compared to miR398-OE (Figure 2A,B). In addition, heat stress caused a significant decrement of RWC and increment of REL and MDA content in both WT and miR398-OE, while more significant heat stress-induced decline of water status and oxidative damage was observed in WT than that in miR398-OE (Figure 2C–E). In conclusion, the results show that miR398-OE was more tolerant to heat stress than WT.

3.3. The Expression of HSFs/HSPs Was Enhanced in miR398-OE

To investigate the mechanisms behind the enhanced heat stress tolerance in miR398-OE, the expression of HSF and HSP genes, including HSFA1A, HSFA2, HSP17.6, HSP70-2, and HSP90-1 were determined in WT and miR398-OE at 1 and 3 HAT. In general, the expression of all the HSFs and HSPs determined in this study was dramatically upregulated in tomato in response to heat stress (Figure 3), while a more significant increment of the expression of HSFA1a, HSFA2, HSP17.6, HSP70-2, and HSP90-1 was observed in miR398-OE under heat stress at both 1 and 3 HAT (except HSP17.6 at 3 HAT) in comparison with that in WT (Figure 3).

3.4. miR398-OE Accumulated More ROS Under Heat Stress than WT

The accumulation of reactive oxygen species (ROS), including superoxide anions (O2·−) and hydrogen peroxide (H2O2) in WT and miR398-OE, was further evaluated using nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) staining and quantitative assays. As shown in Figure 4A, no significant difference in ROS accumulation was determined between WT and miR398-OE when grown without heat stress (0 HAT), while heat stress caused a dramatic accumulation of ROS in tomato shoots. Moreover, more saturated DAB (at 1HAT) and NBT (at 1 and 3 HAT) staining blot was observed in miR398-OE under heat stress compared to WT (Figure 4). In accordance with the results of NBT and DAB staining, the results of quantitative determination of ROS showed that heat stress induced a more significant accumulation of ROS (except the content of H2O2 at 3 HAT) in miR398-OE in comparison with that in WT (Figure 4B,C).

3.5. Overexpression of miR398 Decreased Antioxidant Enzyme Activity in Tomato Under Heat Stress

The activity of SOD, CAT, APX, and GPOD in WT and miR398-OE under heat stress was determined in this study. In general, heat stress promoted the activity of CAT, APX, and GPOD, while the activity of SOD was decreased in WT. Meanwhile, the upregulated expression of CAT, APX, and GPOD was also observed in WT under heat stress (Figure 5). In comparison with the changes of antioxidant enzymes in WT under heat stress, the activity of SOD, CAT, APX, and GPOD, and the expression of CAT, APX, and GPOD was significantly lower in miR398-OE, which was inconsistent with the higher accumulation of ROS in miR398-OE (Figure 5).

3.6. H2O2 Scavenger Treatment Eliminated the Difference of Growth and HSFs/HSPs Expression Between WT and miR398-OE Under Heat Stress

To test the role of ROS accumulation in the miR398-mediated modulation of heat stress tolerance in tomato, dimethylthiourea (DMTU, a H2O2 scavenger) was used in this study. Tomato seedlings of WT and miR398-OE were pretreated with DMTU in the form of foliar spray for 2 d, and then exposed to heat stress for another 2 d. In contrast to the enhanced heat stress tolerance in miR398-OE mentioned above, after the pretreatment with DMTU, no significant difference in shoot biomass, RWC, and MDA content was observed after 2 d of exposure to heat stress (Figure 6). In addition, in accordance with the tomato growth parameters, the expression of HSFs and HSPs in WT and miR398-OE showed no significant difference under heat stress, indicating that DMTU treatment eliminated the enhanced expression of HSFs/HSPs and growth in miR398-OE under heat stress (Figure 7).

4. Discussion

In the past few decades, microRNAs (miRNAs) have gained increasing attention due to their pivotal regulatory roles in the growth and development of plants and stress tolerance [15,17]. As one of the major miRNAs and a key regulator of SOD activity, microRNA398 (miR398) has been shown to be involved in plant responses to multiple biotic and abiotic stresses, including blast disease [44], mosaic virus [23], drought stress [45], salt stress [31], freezing stress [46], cadmium stress [47], and heat stress [24,25]. Although miR398 is relatively conserved in plants, it is worth noting that the expression patterns and roles of miR398 differ with stress types. Even under similar stress conditions, the response of miR398 and its successive regulation on targets differ significantly among plant species [22], which was also confirmed by the results of this study. In tomato, we found that the expression of miR398 was upregulated, and the expression of CSD1/CSD2 and the activity of SOD were downregulated in response to heat stress, which was similar to that in Arabidopsis [24,25], but different from that in Chinese cabbage [26]. To test the role of the upregulated expression of miR398 in tomato thermotolerance, several key growth parameters related to heat stress tolerance in WT and miR398-OE, including biomass, relative water content (RWC), relative electrolyte leakage (REL), and malonaldehyde (MDA) content were further assessed. The results showed that overexpression of miR398 promoted tomato seedling growth and water status and alleviated heat stress-induced oxidative damage, indicating that the upregulated miR398 protected tomato against heat stress.
In agricultural practice, ambient temperature is a key physical parameter influencing plant growth and yield [5]. Therefore, it is not surprising that heat stress would induce detrimental effects on almost all plant life cycle processes, including germination, development, growth, and reproduction [2]. Under the background of global warming, the increasing occurrence of heat stress in agricultural production threatens stable food supply and causes economic loss significantly [5,7]. As sessile organisms, plants have evolved various mechanisms to survive and reproduce under elevated ambient temperatures, while heat shock factors (HSFs) and heat shock proteins (HSPs) are shown to play a crucial role in plant heat stress tolerance [6]. After sensing elevated temperatures, HSFs would be activated, successively enhancing the accumulation of HSPs and plant heat stress resistance [11,12]. In accordance with the growth promotion in miR398-OE, the results of quantitative PCR showed that the expression of several key members of HSF and HSP families was significantly upregulated in miR398-OE in comparison with that in WT, implying the potential regulation of miR398 on HSFs and HSPs in tomato.
To date, the detailed mechanisms of heat stress sensing and the activation of HSF transcription remain unclear. However, it has been found that the accumulation of reactive oxygen species (ROS) plays a key role in modulating HSF transcription and HSP accumulation, thereby participating in plant heat stress acclimation [48]. Considering the regulative nature of miR398 on SOD activity and ROS balance in plants, it can be hypothesized that miR398 may modulate HSF/HSP response and heat stress tolerance via regulating ROS balance. Therefore, the accumulation of ROS in WT and miR398-OE was determined using chemical staining and quantitative assays. The results showed that overexpression of miR398 enhanced the accumulation of ROS in tomato under stress, which could be an underlying mechanism responsible for the upregulated expression of HSFs and HSPs in tomato. Furthermore, the role of ROS in the enhanced heat stress tolerance in miR398-OE was assessed using the pretreatment using DMTU (a ROS scavenger). After 2 d of pretreatment with DMTU and another 2 d of heat stress exposure, no significant differences in tomato seedling growth parameters and expression of HSF and HSP genes between WT and miR398-OE were observed. These results confirmed that overexpression of miR398 promoted heat stress tolerance via enhancing ROS accumulation and HSF/HSP expression.
ROS accumulation is a fundamental symptom in plants under different stress conditions including heat stress, while the ROS accumulation status is largely based on the cooperation of different antioxidant enzymes [49]. Although the upregulated expression of miR398 can downregulate the expression of CSDs and SOD activity in plants, it should be noted that several key enzymes other than SOD, such as CAT, APX, and GPOD, also participate in the scavenging of ROS in plants under stressful conditions. In this study, the activities of several key antioxidant enzymes, besides SOD, and the expression of the related genes were assayed. The results indicate that, in addition to the downregulation of SOD activity, which could be due to the upregulation of miR398 and downregulation of CSD, the activity of CAT, POD, and APX was also downregulated under heat stress in miR398-OE in comparison with that in WT. Considering the fact that miR398 can not modulate the activity of antioxidant enzymes except SOD, the downregulated activity of CAT, POD, and APX in miR398-OE under heat stress could based on the feedback of the change of SOD activity. Overall, these results implied that the decreased activity of SOD, CAT, POD, and APX under heat stress could be responsible for the enhanced accumulation of ROS and the heat stress response based on HSFs and HSPs in miR398-OE. Although miR398 has been proven to be involved in the acclimation to different stresses, the response of miR398 differs among stress types [22]. In plants grown under stressful conditions, the accumulation of ROS could act as signal molecules or induce oxidative damage [50]. In the case of heat stress, the accumulation of ROS and its regulation on HSFs/HSPs could be more important than its toxic effects. Therefore, the expression of miR398 was upregulated to facilitate ROS accumulation, which could be the foundation of the distinct response of miR398 to different stressful conditions.

5. Conclusions

Overall, the role of miR398 in heat stress tolerance in tomato was investigated in this study. The results show that heat stress upregulated the expression of miR398, thereby downregulating the expression of CSDs and SOD activity. Subsequently, the response of miR398 to heat stress facilitated the accumulation of ROS in tomato with the involvement of other antioxidant enzymes besides SOD, which would enhance the HSF/HSP response and protect tomato against heat stress (Figure 8).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15020294/s1, Table S1: The sequence of the primers used in this study.

Author Contributions

Conceptualization, G.Y., Y.H. and Z.Z.; investigation and formal analysis, G.Y., B.L., P.W., S.Z., J.D., S.M., X.Z., T.Y., H.Q. and L.C.; writing—original draft preparation, G.Y., B.L. and Y.X.; writing—review and editing, Y.H. and Z.Z.; project administration, Y.H., Z.Z. and G.Y.; funding acquisition, Y.H. and G.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32372677), the Natural Science Foundation of Zhejiang Province (LQ22C150006), and the Research and Development Fund of Zhejiang A&F University (2021FR037).

Data Availability Statement

All the data are included in this paper.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The expression of miR398 (A), CSD1 (B), CSD2 (C), and SOD activity (D) in tomato under heat stress. Tomato seedlings (cv. Microtom) were grown under heat stress for 3 h. Data are average + SD for four single replicates, with asterisks showing significant differences (p < 0.05). SOD, superoxide dismutase, Pro, protein, U, unit, ns, not significant.
Figure 1. The expression of miR398 (A), CSD1 (B), CSD2 (C), and SOD activity (D) in tomato under heat stress. Tomato seedlings (cv. Microtom) were grown under heat stress for 3 h. Data are average + SD for four single replicates, with asterisks showing significant differences (p < 0.05). SOD, superoxide dismutase, Pro, protein, U, unit, ns, not significant.
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Figure 2. The effects of heat stress on tomato growth (A), shoot dry weight (B), relative water content (C), relative electrolyte leakage (D), and MDA content (E) in WT and miR398-OE. Tomato seedlings of WT and miR398-OE were grown under control (CK) or heat stress (HS) for 2 d. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
Figure 2. The effects of heat stress on tomato growth (A), shoot dry weight (B), relative water content (C), relative electrolyte leakage (D), and MDA content (E) in WT and miR398-OE. Tomato seedlings of WT and miR398-OE were grown under control (CK) or heat stress (HS) for 2 d. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
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Figure 3. Relative expression rates of HSFA1a (A), HSFA2 (B), HSP17.6 (C), HSP70-2 (D), and HSP90-1 (E) in WT and miR398-OE under heat stress. Tomato seedlings of them were cultivated for 3 h under heat stress. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
Figure 3. Relative expression rates of HSFA1a (A), HSFA2 (B), HSP17.6 (C), HSP70-2 (D), and HSP90-1 (E) in WT and miR398-OE under heat stress. Tomato seedlings of them were cultivated for 3 h under heat stress. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
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Figure 4. ROS accumulation in WT and miR398-OE under heat stress. Visualization of superoxide anion (O2·−) and H2O2 with the aid of nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) staining (A), H2O2 content (B), and O2·− content (C). Tomato seedlings of WT and miR398-OE were grown under heat stress for 3 h. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
Figure 4. ROS accumulation in WT and miR398-OE under heat stress. Visualization of superoxide anion (O2·−) and H2O2 with the aid of nitroblue tetrazolium (NBT) and diaminobenzidine (DAB) staining (A), H2O2 content (B), and O2·− content (C). Tomato seedlings of WT and miR398-OE were grown under heat stress for 3 h. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
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Figure 5. The activities of SOD (A), CAT (B), APX (C), GPOD (D), and the expression of CAT (E), APX (F), and GPOD (G) in WT and miR398-OE under heat stress. Tomato seedlings of WT and miR398-OE were grown under heat stress for 3 h. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05). SOD, superoxide dismutase, CAT, catalase, APX, ascorbate peroxidase, GPOD, guaiacol peroxidase, U, unit, Pro, protein, ASA, ascorbate.
Figure 5. The activities of SOD (A), CAT (B), APX (C), GPOD (D), and the expression of CAT (E), APX (F), and GPOD (G) in WT and miR398-OE under heat stress. Tomato seedlings of WT and miR398-OE were grown under heat stress for 3 h. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05). SOD, superoxide dismutase, CAT, catalase, APX, ascorbate peroxidase, GPOD, guaiacol peroxidase, U, unit, Pro, protein, ASA, ascorbate.
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Figure 6. The effects of DMTU pretreatment on shoot fresh weight (A), shoot dry weight (B), relative water content (C), and shoot MDA content (D) in WT and miR398-OE under heat stress. Tomato seedlings of two varieties were pretreated using 10 mM DMTU in the form of foliar spray for 2 d and then grown under control (CK) or heat stress (HS) for another 2 d. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
Figure 6. The effects of DMTU pretreatment on shoot fresh weight (A), shoot dry weight (B), relative water content (C), and shoot MDA content (D) in WT and miR398-OE under heat stress. Tomato seedlings of two varieties were pretreated using 10 mM DMTU in the form of foliar spray for 2 d and then grown under control (CK) or heat stress (HS) for another 2 d. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
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Figure 7. The effects of DMTU pretreatment on the expression of HSFA1a (A), HSFA2 (B), HSP17.6 (C), HSP70-2 (D), and HSP90-1 (E) in WT and miR398-OE under heat stress. Tomato seedlings of WT and miR398-OE were pretreated using 10 mM DMTU in the form of foliar spray for 2 d and then grown under heat stress for another 3 h. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
Figure 7. The effects of DMTU pretreatment on the expression of HSFA1a (A), HSFA2 (B), HSP17.6 (C), HSP70-2 (D), and HSP90-1 (E) in WT and miR398-OE under heat stress. Tomato seedlings of WT and miR398-OE were pretreated using 10 mM DMTU in the form of foliar spray for 2 d and then grown under heat stress for another 3 h. Data are average + SD for four single replicates, with different letters showing significant differences (p < 0.05).
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Figure 8. A schematic model of the function of miR398 in heat acclimation in tomato. The upregulated expression of miR398 would reduce the expression of CSDs and the activity of SOD, thereby promoting ROS accumulation and HSF/HSP expression in tomato under heat stress.
Figure 8. A schematic model of the function of miR398 in heat acclimation in tomato. The upregulated expression of miR398 would reduce the expression of CSDs and the activity of SOD, thereby promoting ROS accumulation and HSF/HSP expression in tomato under heat stress.
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MDPI and ACS Style

Li, B.; Wang, P.; Zhao, S.; Dong, J.; Mao, S.; Zhu, X.; Yuan, T.; Qiu, H.; Cao, L.; Xu, Y.; et al. Sly-miR398 Participates in Heat Stress Tolerance in Tomato by Modulating ROS Accumulation and HSP Response. Agronomy 2025, 15, 294. https://doi.org/10.3390/agronomy15020294

AMA Style

Li B, Wang P, Zhao S, Dong J, Mao S, Zhu X, Yuan T, Qiu H, Cao L, Xu Y, et al. Sly-miR398 Participates in Heat Stress Tolerance in Tomato by Modulating ROS Accumulation and HSP Response. Agronomy. 2025; 15(2):294. https://doi.org/10.3390/agronomy15020294

Chicago/Turabian Style

Li, Baoyu, Peiwen Wang, Shuaijing Zhao, Jiaqi Dong, Shengming Mao, Xuyongjie Zhu, Tiantian Yuan, Haiying Qiu, Long Cao, Yunmin Xu, and et al. 2025. "Sly-miR398 Participates in Heat Stress Tolerance in Tomato by Modulating ROS Accumulation and HSP Response" Agronomy 15, no. 2: 294. https://doi.org/10.3390/agronomy15020294

APA Style

Li, B., Wang, P., Zhao, S., Dong, J., Mao, S., Zhu, X., Yuan, T., Qiu, H., Cao, L., Xu, Y., He, Y., Zhu, Z., & Yan, G. (2025). Sly-miR398 Participates in Heat Stress Tolerance in Tomato by Modulating ROS Accumulation and HSP Response. Agronomy, 15(2), 294. https://doi.org/10.3390/agronomy15020294

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