Introduction

As a result of climate change, observations indicate worldwide increases in temperature and more frequent extreme heat waves. These events pose a threat to plant growth and ecosystems as heat stress damages cell membranes, inactivates enzymes, inhibits photosynthesis, and enhances respiration (Janni et al. 2020; Li et al. 2021; Zhao et al. 2021). The phytohormones, jasmonic acid (JA), and methyl jasmonic acid (MeJA), collectively known as jasmonates, are induced in response to heat stress and act as signaling molecules in stress signaling pathways, enabling plants to adapt to stressful environments (Creelman and Mullet 1995; Clarke et al. 2009; Howe et al. 2018; Wang et al. 2018, 2020; Kanagendran et al. 2019; Su et al. 2021; Nie et al. 2022). The application of exogenous MeJA has demonstrated the role played by MeJA in alleviating heat stress injury in many plant species (Clarke et al. 2009; Pan et al. 2019; Su et al. 2021; Nie et al. 2022). Exogenous MeJA significantly improves heat tolerance in perennial ryegrass (Lolium perenne) by altering osmotic adjustment, antioxidant defenses, and JA-responsive gene expression (Su et al. 2021), and by mediating gene expression across various pathways (Nie et al. 2022). Similarly, application of exogenous JA rescues heat stress-induced stigma secretion (Pan et al. 2019). Clarke et al. (2009) observed that, in Arabidopsis thaliana, exogenous MeJA protects cell membranes from heat stress and improved heat resistance. Moreover, exogenous JA rapidly and dynamically regulates expression of genes involved in plant defense mechanisms under abiotic stress. For example, JA regulates expression of NAC family transcription factors (TFs), which play vital roles in promoting plant adaptations to abiotic stress (Olsen et al. 2005; Jeong et al. 2010; Sun et al. 2013; Zhou et al. 2013; Liang et al. 2014). In addition, upregulation of LpNAC037, LpNAC045, and LpNAC054 occurs in exogenous MeJA-treated leaves of perennial ryegrass under heat stress (Su et al. 2021), upregulation of ethylene response factor (ERF) has been observed in MeJA-treated kiwi fruit under heat stress, and expression of bHLH, MYB, and MPK increases following treatment with exogenous MeJA in various plant species (Yue et al. 2012; Zhao et al. 2013; Pan et al. 2019; Li et al. 2020; Wu et al. 2023). Heat shock proteins (HSPs), controlled by heat stress transcription factors, play a central role in the heat stress response (Kotak et al. 2007; Scharf et al. 2012). Overexpression of Hsp70 improves the tolerance to heat stress of Arabidopsis thaliana (Koizumi et al. 2014), Capsicum annuum (Guo et al. 2014; Usman et al. 2015), and Oryza sativa (Wang et al. 2014). Application of exogenous MeJA under heat stress conditions leads to upregulation of HSP70 and HSP90 in opium poppy (Gurkok et al. 2015) and heat shock factors (Wang et al. 2016) and LpHsp010 (Su et al. 2021) in perennial ryegrass.

Transcriptomic analysis has shown that genes including NAC (AkNAC19 and AkNAC2), MPK (AkMPK6), ERF (AkERF4), EFP (AkEFP), bHLH (AkbHLH), HSP (AkHSP17.6), and MYB (AkMYB123), are upregulated in Abies koreana in response to heat stress (Hwang et al. 2018). Upregulation of these genes in other plant species under heat stress or other abiotic stresses is associated with exogenous JA treatment or endogenous JA synthesis, as described above. It remains unknown, however, whether upregulation of these genes under heat stress is associated with endogenous JA synthesis and signaling in A. koreana, as previous studies have not addressed the effects of exogenous JA on transcriptional regulation during heat stress in this species. We therefore investigated whether MeJA treatment led to the upregulation of these genes in A. koreana. In addition, we evaluated gene expression, electrolyte leakage, and chlorophyll content to determine whether MeJA treatment alleviated the adverse effects of heat stress in Korean fir.

Materials and methods

Plant materials

Plants used in this study were 5-year-old Korean fir (Abies koreana) obtained from the Plant Conservation Center, National Park Institute for Wildlife Conservation, Korea National Park Service. The pots containing seedlings were moved to a growth room within the laboratory and plants were allowed to adapt to growth conditions of 200 µmol/m2 s light intensity, 20 °C temperature, 60% relative humidity, and 16-h photoperiod for 1 month. Plants were then transferred to a plant growth chamber and exposed to a light intensity of 120 µmol/m2 s with other environmental parameters remaining the same.

Treatment of exogenous MeJA

MeJA solutions of 0.1, 1.0, and 2.0 mM were prepared using 0.1% ethanol and 0.05% Tween 20 (Bioshop, Canada, USA). Needles (leaves) of A. koreana plants were dipped in MeJA solution for 1 min. The dipping was performed twice at 5 min intervals. Needles were dipped in distilled water containing 0.05% Tween 20 as a control treatment. During dipping, the pots, but not the plants, were covered with a net to prevent soil loss during inversion. This net was removed after dipping and each plant was covered with a plastic cup for 24 h to prevent evaporation of the MeJA solution. The plants were then returned to the growth chamber. Each treatment was replicated three times and each replicate contained five plants. Samples of needles were collected from MeJA-treated and control plants 24 and 48 h post-treatment and frozen in liquid nitrogen prior to RNA extraction.

RNA extraction and gene expression analysis

Following Avelar Carpinetti et al. (2021), total RNA was extracted from samples with slight modifications. The frozen samples were ground to powder using a bead beater (Retsch MM400, Germany) and 100 mg of the powder was added to 0.9 mL extraction buffer solution [100 mM Tris–HCl (pH 8.0), 10% CTAB, 25 mM EDTA (pH 8.0), 2 M NaCl, 0.02% spermidine trihydrochloride, and 2% β-mercaptoethanol, warmed to 60 ℃]. The samples were mixed well, incubated for 10 min at 60 °C, and 0.9 mL chloroform: isoamyl alcohol (24:1) was added. After centrifugation at 7000 × g for 20 min at 4 °C, 0.6 mL of the supernatant was transferred to a 1.5 mL tube containing 0.9 mL of chloroform: isoamyl alcohol (24:1), and the centrifugation was repeated. Next, the supernatant was transferred to a new tube containing a half volume of 5 M LiCl. The tubes were incubated for 4 h at −20 ℃ and centrifuged at 16,000 × g for 30 min at 4 °C. The RNA pellet was washed three times with 75% ethanol and then dried. The RNA was dissolved in nuclease-free water prior to complementary DNA (cDNA) synthesis.

cDNA was synthesized from 1.0 μg RNA using the ReverTra Ace kit (Toyobo, Japan) according to the manufacturer’s instructions. qRT-PCR analysis was performed as described in Hwang et al. (2018, 2019). The expression levels of AkNAC19, AkMPK6, AkERF4, AkEFP, AkNAC2, AkbHLH, AkHSP17.6, and AkMYB123 were measured in needles relative to the expression of the AkACTIN reference gene using the CFX 96 real-time PCR system (Bio-Rad, USA). Relative gene expression levels were calculated using the quantitative comparative CT (DDCT or ∆∆CT) method. The primers used to amplify the genes of interest (AkNAC19, AkMPK6, AkERF4, AkEFP, AkNAC2, AkbHLH, AkHSP17.6, and AkMYB123) and AkACTIN are listed in Table 1. The analyses were repeated three times for all samples.

Table 1 Primer sequences used for qRT-PCR analyses of gene expression
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Analysis of gene expression at different time points after treatment with exogenous MeJA

Needles of 5-year-old plants were dipped in 2.0 mM MeJA and distilled water containing 0.05% Tween 20 was used as a control treatment. Each treatment was replicated three times and each replicate contained five plants. Samples of MeJA-treated needles were collected 1, 2, 4, and 8 days post-treatment for RNA extraction. RNA was also extracted from control samples 1 day post-treatment. Total RNA extraction, cDNA synthesis, and gene expression analysis were performed as described previously (Hwang et al. 2018).

Treatments of exogenous MeJA and heat stress

Five-year-old plants were dipped in 2.0 mM MeJA solution, as described above and plants dipped in distilled water containing 0.05% Tween 20 were used as untreated controls (no treatment; NT). To determine whether MeJA played a role in alleviating heat stress, plants were placed in growth chambers at either 20 °C (normal condition) or 30 °C (heat stress condition) and 60% RH and 16 h photoperiod. Each treatment was replicated three times and each replicate contained five plants. The electrolyte leakage percentage and chlorophyll content (chlorophyll a and chlorophyll b) were assessed in needles after 28 days of heat stress. In addition, needle samples were collected for RNA extraction after 3 and 28 days of heat stress. Total RNA extraction, cDNA synthesis, and gene expression analysis were performed as described above.

Measurement of electrolyte leakage

To determine electrolyte leakage, needle samples (~3.0 g) were immersed in 30 mL of deionized sterile water in a 50 mL conical tube and maintained in the dark at 25 °C for 24 h before the initial electrical conductivities (EC1 and W1) were evaluated. The conical tube was then incubated at 121 °C for 5 min and cooled to 25 °C prior to measurement of the final electrical conductivities (EC2, W2).

Electrolyte leakages (EL) were calculated using the following equation (Binder and Fielder 1995):

$$\text{EL}(\%) = (\text{EC}1 - \text{W}1 )\times 100 \slash {(\text{EC}2 - \text{W}2)}.$$

Measurement of chlorophyll content

Fresh needles, frozen in liquid nitrogen, were ground into powder (100 mg each) for total chlorophyll extraction, 1.0 mL of 80% Aceton (aceton:DW 8:2, v/v) (Sigma-Aldrich, USA) was added, and the samples were mixed well prior to centrifugation at 13,000 rpm for 5 min at 4 ℃. The supernatants were transferred to 96-well plates and their optical density was measured at 663 and 646 nm. The measurement was repeated three times. Chlorophyll a and chlorophyll b contents were calculated using the following equations (Lichtenthaler and Wellburn 1983):

$${\text{Chlorophyll a }} ({\text{mg}} \slash {\text{mL}} ) = 12.21{\text{A}}_{663} - 2.81 {\text{A}}_{646},$$
$$ {\text{Chlorophyll b }} ({\text{mg}} \slash {\text{mL}} )= {20.13\text{A}}_{646}-{5.03\text{A}}_{663}.$$

Results

Role of exogenous MeJA in regulation of stress-responsive genes

Transcriptional upregulation in response to heat stress of the stress-responsive genes (AkNAC19, AkMPK6, AkERF4, AkEFP, AkNAC2, AkbHLH, AkHSP17.6, and AkMYB123) has been observed in A. koreana (Hwang et al. 2018). To investigate whether these genes were also upregulated following application of exogenous MeJA, A. koreana needles were treated with different concentrations of MeJA. Needles were harvested 24 and 48 h after treatment for analysis of gene expression levels. Gene expression in plants treated with 0.1 mM MeJA did not differ significantly from that in the control group at either 24 h or 48 h post-treatment (Fig. 1). Gene expression was noticeably upregulated following treatment with higher concentrations of MeJA (1.0 and 2.0 mM). However, although the expression levels of AkNAC19, AkERF4, AkEFP, AkNAC2, AkbHLH and AkHSP17.6 were highest 24 h after treatment with 2.0 mM MeJA. AkMPK6 expression did not differ significantly between these treatments (Fig. 1). The expression levels of most genes, other than AkNAC2 and AkHSP17.6, had declined by 48 h post-treatment, particularly following treatment with 2.0 mM MeJA. Upregulation of genes other than AkHSP17.6 was not observed 48 h post-treatment with other MeJA concentrations. Overall, upregulation of gene expression was associated with MeJA application, although the expression level varied with concentration and time point. As six out of the eight genes were strongly upregulated 24 h after treatment with 2.0 mM MeJA, this concentration was selected for further experiments.

Fig. 1
figure 1

Expression levels of the investigated genes in A. koreana needles 24 and 48 h post-treatment with different concentrations of MeJA. Data represent means of three replicates. The error bars show standard error of the mean. Statistically significant differences relative to each control of 24 and 48 h post-treatment are indicated using asterisks (*p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test). Con: distilled water-treated control

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Next, needles were treated with either 2.0 mM MeJA or distilled water containing 0.05% Tween 20 as a control. Gene expression levels in MeJA-treated needles were assessed at 1, 2, 4, and 8 days post-treatment and in control needles after 1 day. Gene expression was higher in MeJA-treated needles than in the control group, although the level of expression varied across time points (Fig. 2). Expression of all genes other than AkHSP17.6 was highest at 1 day post-treatment, consistent with the results of the first experiment. A strong or slight decline in expression of AkNAC19, AkERF4, AkEFP, AkNAC2, and AkMYB123 was observed over time. By contrast, expression of AkMPK6, AkbHLH, and AkHSP17.6 did not depend on time point. AkbHLH expression in treated needles at 2, 4, and 8 days post-treatment was lower than in the control and, at 4 days post-treatment, AkHSP17.6 expression in treated needles was also lower than in the control, although expression of this gene peaked 8 days post-treatment. These results suggested that treatment with 2.0 mM MeJA was associated with strongly upregulated expression of the selected genes in A. koreana, particularly during the first day with expression levels declining thereafter.

Fig. 2
figure 2

Expression of the investigated genes in A. koreana needles 1, 2, 4, and 8 days post-treatment with 2.0 mM MeJA. Data represent means of three replicates. The error bars show standard error of the mean. Statistically significant differences relative to control are indicated using asterisks (*p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test). Con: distilled water-treated control

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Role of exogenous MeJA in alleviating heat stress

To determine whether treatment with exogenous MeJA alleviated the adverse effects of heat stress in A. koreana, plants treated with 2.0 mM MeJA (MJ) and untreated controls (NT) were grown for 28 days in growth chambers set either at 30 °C to induce heat stress or at 20 °C to provide a non-stressed environment (Fig. 3a). In addition, EL and chlorophyll content both play vital roles in the adaptation of plants to heat stress. EL in the needles was assessed after 28 days. Similar EL percentages were observed in the MeJA-treated and NT plants under non-stress conditions. Under heat stress conditions, however, although both treated and NT plants showed significantly elevated EL, the percentage of EL in NT plants was significantly higher than in MeJA-treated plants (Fig. 3b).

Fig. 3
figure 3

Comparison of changes in A. koreana needles treated with 2.0 mM MeJA (MJ) or distilled water (NT) after exposure to 28 days of non-stress (20 °C) or heat stress (30 °C) conditions. a Phenotypes of A. koreana treated with 2.0 mM MeJA (MJ) or distilled water (NT) after exposure to 28 days of non-stress (20 °C) or heat stress (30 °C) conditions. b Electrolyte leakage assay. c, d Analysis of chlorophyll contents. Data represent means of three replicates. The error bars show standard error of the mean. Statistically significant differences relative to controls (20 °C or 30 °C) are indicated using asterisks [*p < 0.05, **p < 0.01, ***p < 0.001 (20 °C) and ##p < 0.01, ###p < 0.001 (30 °C); Student’s t test]

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In addition, the chlorophyll content [chlorophyll a (Chl a) and chlorophyll b (Chl b) was measured after 28 days. At 20 °C, the Chl a content in NT needles was slightly higher than that in MeJA-treated needles, whereas the opposite result was observed for Chl b. Although a significant decline in chlorophyll content was observed in both groups at 30 °C, levels of Chl a and Chl b in MeJA-treated needles were significantly or slightly higher than those in NT needles (Fig. 3c, d). These results suggested that MeJA treatment alleviated the adverse effects of heat stress in A. koreana by reducing the EL percentage and maintaining the chlorophyll content.

Expression of stress-responsive genes in MeJA-treated needles under heat stress

The previous experiment showed that MeJA treatment reduced the adverse effects of heat stress in A. koreana. We, therefore, evaluated levels of expression of stress-responsive genes in MeJA-treated and NT needles after 3 and 28 days of heat stress (30 °C), respectively (Figs. 4, 5). After 3 days in unstressed conditions (20 °C), the expression levels of all genes except AkNAC19 were similar in both MeJA-treated and NT needles; AkNAC19 expression was higher in MeJA-treated needles than in NT needles. At 30 °C, however, we observed that expression of all genes except AkMPK6 was upregulated after 3 days. Most genes were expressed at significantly higher levels in MeJA-treated needles than in NT needles after 3 days under heat stress conditions; the exception was AkEFP, which was expressed at a higher level in NT needles than in MeJA-treated needles (Fig. 4).

Fig. 4
figure 4

Expression levels of the investigated genes in A. koreana needles treated with 2.0 mM MeJA (MJ) or distilled water (NT) after exposure to 3 days of non-stress (20 °C) or heat stress (30 °C) conditions. Data represent means of three replicates. The error bars show standard error of the mean. Statistically significant differences relative to controls (20 °C or 30 °C) are indicated using asterisks [*p < 0.05, **p < 0.01 (20 °C) and #p < 0.05, ##p < 0.01, ###p < 0.001 (30 °C); Student’s t test]

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Fig. 5
figure 5

Expression levels of the investigated genes in A. koreana needles treated with 2.0 mM MeJA (MJ) or distilled water (NT) after exposure to 28 days of non-stress (20 °C) or heat stress (30 °C) conditions. Data represent means of three replicates. The error bars show standard error of the mean. Statistically significant differences relative to controls (20 °C or 30 °C) are indicated using asterisks [*p < 0.05, **p < 0.01, ***p < 0.001 (20 °C) and #p < 0.05, ##p < 0.01, ###p < 0.001 (30 °C); Student’s t test]

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After 28 days at 20 °C, the expression levels of all genes except AkNAC19 and AkbHLH were similar in MeJA-treated and NT needles. The result resembled that obtained after 3 days. After 28 days at 30 °C, however, AkERF4, AkNAC2, and AkHSP17.6 showed elevated levels of expression, and expression of these genes was significantly higher in MeJA-treated needles than in NT needles. By contrast, after 28 days of heat stress, expression of AkEFP and AkMYB123 was strongly elevated in NT needles to levels that were significantly higher than in MeJA-treated needles (Fig. 5). Overall, the genes investigated in this study were upregulated under heat stress conditions in MeJA-treated and NT plants. Upregulation of AkERF4, AkNAC2, and AkHSP17.6 was observed in MeJA-treated and NT needles soon after the onset of heat stress (day 3) as well as after an extended period of heat stress (day 28), although expression levels were higher at the earlier time point. This suggested that application of exogenous MeJA strongly upregulated expression of most genes throughout the duration of heat stress conditions.

Discussion

MeJA production is induced in plants exposed to heat stress as MeJA plays a role in enabling plants to adapt to heat stress conditions (Clarke et al. 2009; Howe et al. 2018; Wang et al. 2018, 2020; Kanagendran et al. 2019; Su et al. 2021; Nie et al. 2022). Hwang et al. (2018) reported that eight stress-induced genes (AkNAC19, AkMPK6, AkERF4, AkEFP, AkNAC2, AkbHLH, AkHSP17.6, and AkMYB123) were induced in A. koreana in response to heat stress and involved in the heat stress-tolerance mechanism. It remained unclear, however, whether these stress-induced genes were associated with MeJA production. We, therefore, designed the present study to investigate whether application of exogenous MeJA led to upregulation of these eight genes and to assess the role of MeJA in alleviating the deleterious effects of heat stress in A. koreana.

Here, treatment with MeJA led to upregulated expression of the stress-induced genes. Treatment with 0.1 mM MeJA was insufficient to upregulate gene expression, however, as no difference was observed between plants treated with this low concentration and untreated controls. Significant upregulation of gene expression was observed in plants treated with 1.0 or 2.0 mM MeJA with the higher concentration producing the greater effect (Fig. 1). Shahzad et al. (2015) reported that 0.1 mM or 0.2 mM MeJA enabled pea plants to adapt to heat stress and that high concentrations of MeJA promoted plant defense mechanisms, enabling adaptation to high temperature (40 °C) stress. Su et al. (2021) observed that treatment with 0.1 mM MeJA produced the most favorable effect on the heat tolerance of perennial ryegrass. This may be because MeJA causes strong upregulation of stress-responsive genes, thereby alleviating the deleterious effects of heat stress on plant growth. Rahman et al. (2022) observed that treatment of grapevine leaves with 100 µM MeJA led to significant upregulation of genes involved in metabolic pathways and stress-tolerance mechanisms. Treatment with 100 µM MeJA was associated with increased expression of MYB4 and MYB88 in Glycyrrhiza uralensis (Li et al. 2020), and ERF TFs in California poppy (Yamada et al. 2020) and tomato fruit (Yu et al. 2018). In addition, strong upregulation of the MPK was observed in Populus trichocarpa exposed to 0.5 mM MeJA (Wu et al. 2023).

The concentration of MeJA required to upregulate stress-induced gene expression in A. koreana differed from those used in the studies cited above, as did plant responses to stress. Although treatment with 0.1 mM MeJA led to upregulated gene expression in other plants, up to 2.0 mM MeJA was required in A. koreana. This may be because the physiological status of the plants, as well as the level of endogenous MeJA, differed between studies and species. In addition, the needles of A. koreana are thicker and more rigid in structure and texture than the leaves of the plant species studied previously, which may limit the effective absorption when needles are treated with low concentrations of MeJA. We found that the level of upregulation of gene expression following MeJA treatment depended on the time of testing, as the expression levels of most genes peaked at 24 h post-treatment and had declined by 48 h (Fig. 1). It is likely that MeJA upregulated most genes early in the post-treatment period. Rahman et al (2022) observed that the flavonoid genes F3H and PAL were strongly upregulated 24 h after MeJA treatment, but their expression levels declined by 48 h. Gurkok et al. (2015) also reported that HSP70 and HSP90 were upregulated in opium poppy 3 and 12 h after MeJA treatment. To validate our results, levels of gene expression were measured in A. koreana needles 0, 1, 2, 4, and 8 days after treatment with 2.0 mM MeJA (Fig. 2). Consistent with earlier studies, expression of most genes was highest 1 day (24 h) after treatment, confirming that gene expression in A. koreana was strongly upregulated by exogenous MeJA early in the post-treatment period.

EL and chlorophyll content indicate the level of injury caused to plants by abiotic stress. Higher EL percentages and decreased chlorophyll content are both negatively associated with plant growth. An elevated EL percentage and a decline in chlorophyll content were observed in both MeJA-treated and NT plants under heat stress in this study. Such heat stress-induced damage has been previously reported in many plant species (Zhou and Leul 1999; Hu et al. 2020; Liu and Huang 2000; Su et al. 2021). Treatment with 2.0 mM MeJA alleviated the deleterious effects of heat stress in A. koreana, as lower EL percentages were observed under heat stress conditions in MeJA-treated plants than in NT plants (Fig. 3b). Similarly, the chlorophyll content in MeJA-treated plants was higher than that in NT plants (Fig. 3c, d). Such MeJA-mediated alleviation of heat stress injuries has been reported previously (Hu et al. 2013; Shahzad et al. 2015; Bertini et al. 2019; Su et al. 2021; Nie et al. 2022). MeJA protects plants from photosynthetic damage by maintaining chlorophyll content (Bertini et al. 2019) and by increasing expression of chlorophyll biosynthesis genes under heat stress (Nie et al. 2022). Su et al (2021) reported that MeJA could maintain chlorophyll content and decrease EL in perennial ryegrass leaves under heat stress. In addition, MeJA-associated reduction in EL has been observed in Arabidopsis thaliana and pea plants under heat stress (Hu et al. 2013; Shahzad et al. 2015). An increase in chlorophyll and carotenoid contents, soluble proteins, flavonoids, lignin, and enzymatic antioxidants, which play a crucial role in stress tolerance, was also recently reported in MeJA-treated leaves of Isatis indigotica (Liu et al. 2022).

To clarify whether MeJA-mediated heat stress alleviation was associated with expression of AkNAC19, AkMPK6, AkERF4, AkEFP, AkNAC2, AkbHLH, AkHSP17.6, and AkMYB123, the expression levels of these genes were assessed in MeJA-treated and NT needles under heat stress (30 °C) and non-stress (20 °C) conditions (Figs. 4, 5). All genes except AkMPK6 showed upregulated expression after 3 days of heat stress. Moreover, gene expression levels in MeJA-treated needles were higher than those in NT leaves. These results indicated not only that these genes were involved in heat stress tolerance but also that their expression was upregulated by MeJA (Fig. 4). After exposure to heat stress for 28 days, we observed elevated expression of AkERF4, AkNAC2, and AkHSP17.6. Again, expression of these genes was significantly higher in the MeJA-treated needles than in NT needles. Expression levels of AkNAC19 and AkMPK6 decreased in both MeJA-treated and NT needles (Fig. 5). Therefore, the greater tolerance of heat stress shown by MeJA-treated plants may result from upregulated expression of AkERF4, AkNAC2, and AkHSP17.6, both early and late in the period of exposure to heat stress. These three genes are strong candidates for genes that act to alleviate the deleterious effects of heat stress. In addition, AkMPK6 may be involved in the stress-tolerance mechanism at the post-translational modification, as its expression was not upregulated under heat stress conditions in either MeJA-treated or NT needles.

Heat stress-induced upregulation of NAC TFs has been observed in A. koreana, Arabidopsis thaliana, and ryegrass (Morishita et al. 2009; Hwang et al. 2018; Su et al. 2021). Su et al (2021) reported that upregulation of three NAC TFs, LpNAC037, LpNAC045, and LpNAC054, by MeJA enabled ryegrass to tolerate heat stress. Similarly, upregulation of ERF genes during heat stress has been observed in A. koreana, Arabidopsis thaliana, and pak choi (Hsieh et al. 2013; Xu et al. 2016; Hwang et al. 2018). Increased expression of ERF genes in MeJA-treated leaves is consistent with other studies. For instance, Yamada et al. (2020) and Yu et al. (2018) observed that MeJA strongly induced expression of several ERF genes in California poppy and tomato fruit, respectively. More recently, Nie et al. (2022) and Su et al. (2021) have shown that improved tolerance of ryegrass to heat stress is associated with MeJA-induced upregulation of HSP genes. In the current study, 2.0 mM MeJA was required to upregulate genes involved in the stress-tolerance mechanisms of A. koreana. Application of MeJA at this concentration improved plants’ tolerance of heat stress by reducing EL and maintaining chlorophyll content. The beneficial effects of MeJA treatment were strongly associated with upregulation of gene expression, mostly notably of AkERF4, AkNAC2, and AkHSP17.6.

Conclusion

In this study, treatment with exogenous MeJA led to upregulated expression of eight genes (AkNAC19, AkMPK6, AkERF4, AkEFP, AkNAC2, AkbHLH, AkHSP17.6, and AkMYB123) involved in stress-tolerance mechanisms in Abies koreana. A high concentration (2.0 mM) of MeJA was required to upregulate these genes significantly. In addition, the effect of MeJA on gene expression varied over time post-treatment. Expression levels of most genes were highest 1 day post-treatment. Abiotic stresses such as heat shock damage plant cells through increasing EL percentage and reducing chlorophyll content. MeJA treatment alleviated the adverse effects of heat stress on A. koreana by reducing EL and maintaining chlorophyll content. These effects were strongly associated with higher levels of expression of AkERF4, AkNAC2, and AkHSP17.6 in MeJA-treated plants than in the NT plants. This suggested that the ability of A. koreana to tolerate heat stress may be associated with endogenous MeJA synthesis or signaling pathways. In addition, AkERF4, AkNAC2, and AkHSP17.6 are potential candidates for target genes for manipulation to improve the tolerance of A. koreana to heat stress, although further studies are required to validate the functions of these genes in abiotic stress tolerance in this species.