Jasmonate Positively Regulates Cold Tolerance by Promoting ABA Biosynthesis in Tomato

As a cold-sensitive species, tomato is frequently challenged by cold stress during vegetative and reproductive growth. Understanding how tomato responds to cold stress is of critical importance for sustainable tomato production. In this work, we demonstrate that jasmonate (JA) plays a crucial role in tomato response to cold stress by promoting abscisic acid (ABA) biosynthesis. It was observed that both JA and ABA levels were substantially increased under cold conditions, whereas the suppression of JA biosynthesis abated ABA accumulation. The ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE2 (NCED2) was subsequently found to be associated with JA-mediated ABA biosynthesis in tomato plants in response to cold stress. NCED2 was rapidly induced by exogenous MeJA and cold treatment. Silencing NCED2 led to a decrease in ABA accumulation that was concurrent with increased cold sensitivity. Moreover, blocking ABA biosynthesis using a chemical inhibitor impaired JA-induced cold tolerance in tomato. Furthermore, MYC2, a core component of the JA signaling pathway, promoted the transcription of NCED2, ABA accumulation and cold tolerance in tomato. Collectively, our results support that JA signaling promotes ABA biosynthesis to confer cold tolerance in tomato.


Introduction
Tomato (Solanum lycopersicum) is well known as an economically important horticultural crop across the globe. Tomato fruits are well recognized as a source of multiple health-promoting nutrients, including lycopene and vitamins [1]. However, during tomato production, abiotic stresses, such as cold, heat, drought and salinity, often inhibit growth and development [2][3][4][5][6]. Cold is a major stress factor that poses great threat to growth, yields and quality in tomato, as this species originates from tropical or subtropical regions [7,8]. Thus, understanding how tomato responds to low-temperature conditions is a subject of substantial interest. A large number of studies have documented the deleterious effects of cold stress in plants. Reactive oxygen species (ROS) are notoriously accumulated under cold stress, eventually leading to oxidative damage to a plethora of macromolecules, such as proteins, nucleic acids and sugars [9][10][11][12]. Cold stress also impairs photosynthesis by inactivating major enzymes that participate in the process of carbon fixation [13]. For example, sedoheptulose-1,7-bisphosphatase, which is a crucial enzyme for the production of ribulose-1,5-bisphosphate in carbon fixation, undergoes a rapid loss of activity under cold conditions [14].
As sessile organisms, plants have evolved sophisticated mechanisms at the physiological, cellular, and molecular levels to cope with low-temperature conditions. At the physiological level, for instance, upon cold stress, plants synthesize and accumulate numerous protective substances and proteins, such as soluble sugars, proline, and LEA proteins [15][16][17]. At the molecular level, several critical genes play a role in the cold response, among which CBF genes are best characterized and play central roles in the transcriptional regulatory network under low-temperature conditions [18][19][20]. CBF transcription

JA and ABA Accumulate in Tomato Leaves in Response to Cold Stress
To understand the potential crosstalk between JA and ABA in tomato plants exposed to cold stress, we first investigated the levels of JA and ABA. It was observed that at 12 h and 24 h following cold treatment, the accumulation of JA was increased by 580% and 310%, respectively ( Figure 1A). Similarly, ABA production was substantially increased by cold stress, with the ABA level being enhanced by 318.0% at 12 h and 220% at 24 h after cold stress was imposed ( Figure 1B). Intriguingly, under cold stress, MeJA substantially elevated the accumulation of ABA in comparison with the mock treatment, whereas sodium diethyldithiocarbamate (DIECA), a JA biosynthesis inhibitor, attenuated cold-triggered  Figure 1C). These observations imply that both JA and ABA may be involved in the tomato cold response and JA may interact with ABA by modulating its accumulation under cold stress. cold stress was imposed ( Figure 1B). Intriguingly, under cold stress, MeJA substa elevated the accumulation of ABA in comparison with the mock treatment, where dium diethyldithiocarbamate (DIECA), a JA biosynthesis inhibitor, attenuated cold gered accumulation of ABA ( Figure 1C). These observations imply that both JA and may be involved in the tomato cold response and JA may interact with ABA by mo ing its accumulation under cold stress. Leaf samples collected at 12 h an following cold stress were used for determination of accumulations of endogenous JA and Exogenous MeJA and DIECA were applied 12 h prior to cold treatment and ABA levels were mined following 24 h of cold stress. Data presented are means of three biological replicates Asterisks indicate significant differences (** p < 0.01, * p < 0.05) according to Student's t-te fresh weight; MeJA, methyl jasmonate; DIECA, sodium diethyldithiocarbamate.

Identification of JA-Responsive ABA Biosynthesis Gene NCED2 under Cold Stress
After observing that JA was involved in ABA accumulation under cold stre next attempted to identify JA-targeted ABA biosynthesis genes in tomato. By ana previously published transcriptome data, we identified a JA-responsive gene N which is known to play a key role in tomato ABA biosynthesis. Sequence analys played that the transcript length of NCED2 was 1746 bp, which encoded a predicte tein of 581 amino acids. Gene structural analysis revealed that NCED2 contained on exon. NCED2 has an estimated molecular weight of 64.83 kDa and a predicted isoe point of 6.51.
We then examined the time course of NCED2 expression in response to MeJA t ify the association of JA with NCED2. Tomato leaves were detached and incuba MeJA solution for 24 h and the relative expression of NCED2 was assessed by qRT During MeJA treatment, NCED2 expression was rapidly induced at 3 h and peaked h ( Figure 2A). After establishing that JA induced the expression of NCED2, we next whether cold stress also triggered NCED2 expression. To seek an answer, we sub tomato plants (four-leaf stage) to cold stress (4 °C) for 24 h. It was shown that cold led to a remarkable increase in the expression of NCED2, with the transcript level increased by 5.9-fold at 5 h following cold treatment ( Figure 2B). These results su that NCED2 is responsive to both JA and cold stress. Leaf samples collected at 12 h and 24 h following cold stress were used for determination of accumulations of endogenous JA and ABA. Exogenous MeJA and DIECA were applied 12 h prior to cold treatment and ABA levels were determined following 24 h of cold stress. Data presented are means of three biological replicates (±SD). Asterisks indicate significant differences (** p < 0.01, * p < 0.05) according to Student's t-test. FW, fresh weight; MeJA, methyl jasmonate; DIECA, sodium diethyldithiocarbamate.

Identification of JA-Responsive ABA Biosynthesis Gene NCED2 under Cold Stress
After observing that JA was involved in ABA accumulation under cold stress, we next attempted to identify JA-targeted ABA biosynthesis genes in tomato. By analyzing previously published transcriptome data, we identified a JA-responsive gene NCED2, which is known to play a key role in tomato ABA biosynthesis. Sequence analysis displayed that the transcript length of NCED2 was 1746 bp, which encoded a predicted protein of 581 amino acids. Gene structural analysis revealed that NCED2 contained only one exon. NCED2 has an estimated molecular weight of 64.83 kDa and a predicted isoelectric point of 6.51.
We then examined the time course of NCED2 expression in response to MeJA to verify the association of JA with NCED2. Tomato leaves were detached and incubated in MeJA solution for 24 h and the relative expression of NCED2 was assessed by qRT-PCR. During MeJA treatment, NCED2 expression was rapidly induced at 3 h and peaked at 12 h ( Figure 2A). After establishing that JA induced the expression of NCED2, we next asked whether cold stress also triggered NCED2 expression. To seek an answer, we subjected tomato plants (four-leaf stage) to cold stress (4 • C) for 24 h. It was shown that cold stress led to a remarkable increase in the expression of NCED2, with the transcript level being increased by 5.9-fold at 5 h following cold treatment ( Figure 2B). These results suggest that NCED2 is responsive to both JA and cold stress. To further verify the relationship between JA and ABA biosynthesis genes sponse to cold stress, we performed pharmacological studies using exogenous MeJ DIECA, which is a commonly used inhibitor of JA biosynthesis. qRT-PCR analy vealed that under cold stress, the application of MeJA markedly promoted the expr of NCED2, while DIECA suppressed the expression of NCED2, compared with the treatment ( Figure 2C). Under optimal growth conditions, MeJA enhanced the trans of NCED2, whereas DIECA did not significantly alter the transcripts of NCED2 in parison with the mock treatment. These results were consistent with the ABA quan tion following treatments of MeJA and DIECA under cold stress ( Figure 1C), sugg that JA promotes ABA accumulation likely by inducing the expression of NCED2 mato challenged by cold stress. Tomato plants at the 4-leaf stage were subjected to cold stress at 4 • C for 24 h and leaf samples were collected at indicated time points for the expression analysis of NCED2 by qRT-PCR. Exogenous MeJA and DIECA were applied to tomato leaves 12 h prior to the cold treatment and the expression of NCED2 was determined by qRT-PCR following 24 h of cold stress. Data presented are means of three biological replicates (±SD). Asterisks indicate significant differences (** p < 0.01, * p < 0.05) according to Student's t-test.

Suppression of NCED2 Attenuates Cold Tolerance in Tomato
To further verify the relationship between JA and ABA biosynthesis genes in response to cold stress, we performed pharmacological studies using exogenous MeJA and DIECA, which is a commonly used inhibitor of JA biosynthesis. qRT-PCR analysis revealed that under cold stress, the application of MeJA markedly promoted the expression of NCED2, while DIECA suppressed the expression of NCED2, compared with the mock treatment ( Figure 2C). Under optimal growth conditions, MeJA enhanced the transcripts of NCED2, whereas DIECA did not significantly alter the transcripts of NCED2 in comparison with the mock treatment. These results were consistent with the ABA quantification following treatments of MeJA and DIECA under cold stress ( Figure 1C), suggesting that JA promotes ABA accumulation likely by inducing the expression of NCED2 in tomato challenged by cold stress.

Suppression of NCED2 Attenuates Cold Tolerance in Tomato
As NCED2 was associated with the tomato cold response, we continued to explore how it might affect tomato cold tolerance. We suppressed the expression of NCED2 via virusinduced gene silencing (VIGS) and plants with reductions in NCED2 transcript abundance by~80% were selected for analyses of ABA levels and cold tolerance ( Figure 3A). Under cold stress, NCED2-silenced plants accumulated remarkably less ABA than the control plants, while under optimal growth conditions, NCED2-silenced plants did not show any significant differences compared to the control plants in the accumulation of ABA ( Figure 3B). Phenotype analysis showed that VIGS plants displayed more cold damage than the control plants ( Figure 3C). Moreover, silencing NCED2 led to a decrease in Fv/Fm ( Figure 3D), which is a commonly used indicator of cold tolerance. Additionally, NCED2silenced plants demonstrated higher relative electrolyte leakage (REL) than the control plants ( Figure 3D). These observations imply that NCED2 positively regulates cold tolerance by increasing ABA accumulation in tomato plants under cold stress.
Plants 2023, 12, x FOR PEER REVIEW abundance by ~80% were selected for analyses of ABA levels and cold tolerance (F 3A). Under cold stress, NCED2-silenced plants accumulated remarkably less ABA the control plants, while under optimal growth conditions, NCED2-silenced plan not show any significant differences compared to the control plants in the accumu of ABA ( Figure 3B). Phenotype analysis showed that VIGS plants displayed mor damage than the control plants ( Figure 3C). Moreover, silencing NCED2 led to a de in Fv/Fm ( Figure 3D), which is a commonly used indicator of cold tolerance. Additio NCED2-silenced plants demonstrated higher relative electrolyte leakage (REL) th control plants ( Figure 3D). These observations imply that NCED2 positively regulate tolerance by increasing ABA accumulation in tomato plants under cold stress. . TRV and TRV-N tomato plants were exposed to cold stress and leaves were collected for the expression anal NCED2 and cold tolerance assays. Data presented are means of three biological replicates Asterisks indicate significant differences (** p < 0.01, * p < 0.05) according to Student's t-test.

ABA Mediates JA-Induced Cold Tolerance
We found that JA induced the expression of NCED2 and NCED2 positively regu cold tolerance by increasing ABA accumulation, which prompted us to hypothesiz ABA mediates JA-induced cold tolerance in tomato. To test this hypothesis, we too vantage of the ABA biosynthesis inhibitor fluridone (Flu) to partially block ABA acc lation in tomato leaves. It was shown that cold stress adversely affected the maxima . TRV and TRV-NCED2 tomato plants were exposed to cold stress and leaves were collected for the expression analysis of NCED2 and cold tolerance assays. Data presented are means of three biological replicates (±SD). Asterisks indicate significant differences (** p < 0.01, * p < 0.05) according to Student's t-test.

ABA Mediates JA-Induced Cold Tolerance
We found that JA induced the expression of NCED2 and NCED2 positively regulated cold tolerance by increasing ABA accumulation, which prompted us to hypothesize that ABA mediates JA-induced cold tolerance in tomato. To test this hypothesis, we took advan- tage of the ABA biosynthesis inhibitor fluridone (Flu) to partially block ABA accumulation in tomato leaves. It was shown that cold stress adversely affected the maximal photochemical efficiency of PSII, as reflected by the substantial decrease in Fv/Fm, while exogenously applied MeJA considerably alleviated cold-induced suppression of Fv/Fm. MeJA-treated plants displayed a higher maximal photochemical efficiency of PSII than the mock plants under cold conditions, with Fv/Fm being increased by 58.8%. However, the application of Flu attenuated the effects of MeJA on Fv/Fm. Compared with the MeJA treatment, the Flu treatment decreased Fv/Fm by 21.6% ( Figure 4A). The assessment of relative electrolyte leakage revealed that under cold stress, MeJA significantly reduced the relative electrolyte leakage, while the addition of Flu weakened the effects of MeJA, leading to increased electrolyte leakage ( Figure 4B). The results demonstrate that JA promotes cold tolerance, depending partially on ABA.
Plants 2023, 12, x FOR PEER REVIEW relative electrolyte leakage revealed that under cold stress, MeJA significantly re the relative electrolyte leakage, while the addition of Flu weakened the effects of leading to increased electrolyte leakage ( Figure 4B). The results demonstrate that J motes cold tolerance, depending partially on ABA.

JA Promotes ABA Biosynthesis via MYC2-Regulated Transcription of NCED2 under Stress
As MYC2 acts as a crucial regulator of the JA signaling pathway, we next exa if JA-induced NCED2 expression and ABA accumulation were associated with MYC utilized previously generated MYC2-RNAi plants to investigate the function of MY the transcription of NCED2 under cold stress. It was observed that under cold cond the suppression of MYC2 led to a decrease in the relative expression of NCED2 by and 31.9% in transgenic lines 2# and 16#, respectively ( Figure 5A). Concomitantly, cumulations of ABA in MYC2-RNAi lines were significantly reduced following cold ( Figure 5B). The cold tolerance assay showed that suppression of MYC2 resulted As MYC2 acts as a crucial regulator of the JA signaling pathway, we next examined if JA-induced NCED2 expression and ABA accumulation were associated with MYC2. We utilized previously generated MYC2-RNAi plants to investigate the function of MYC2 in the transcription of NCED2 under cold stress. It was observed that under cold conditions, the suppression of MYC2 led to a decrease in the relative expression of NCED2 by 36.2% and 31.9% in transgenic lines 2# and 16#, respectively ( Figure 5A). Concomitantly, the accumulations of ABA in MYC2-RNAi lines were significantly reduced following cold stress ( Figure 5B). The cold tolerance assay showed that suppression of MYC2 resulted in decreased cold tolerance, as evidenced by the cold-sensitive phenotype and enhanced relative electrolyte leakage ( Figure 5C,D). In an attempt to further unravel the regulatory role of MYC2 in the transcrip NCED2, we took a closer look at the NCED2 promoter. We analyzed the 2k promo gion using PlantCare [50] and identified two G-box elements that have been repor MYC2 binding sites ( Figure 5E), which are suggestive of the possible association of with the NCED2 promoter. Furthermore, we performed dual-luciferase assays to the transactivation activity of MYC2 on the NCED2 promoter. It was shown that activated the expression of NCED2, which was inferred from the increased promo tivity, as indicated by the enhanced LUC/REN ( Figure 5F). These results clearly d strate that MYC2 positively regulates the transcription of NCED2 and ABA accumu in tomato cold stress.

Discussion
As a major abiotic stress, low temperature inhibits plant growth and causes c erable losses of crop yields. Tomato is a warm-climate species and cold stress adv affects its growth and development at nearly all stages, including vegetative, rep tive, and fruiting. It is, thus, critical to understand how tomato responds to cold st the physiological and molecular levels. Initially identified as a defense hormone a In an attempt to further unravel the regulatory role of MYC2 in the transcription of NCED2, we took a closer look at the NCED2 promoter. We analyzed the 2k promoter region using PlantCare [50] and identified two G-box elements that have been reported as MYC2 binding sites ( Figure 5E), which are suggestive of the possible association of MYC2 with the NCED2 promoter. Furthermore, we performed dual-luciferase assays to verify the transactivation activity of MYC2 on the NCED2 promoter. It was shown that MYC2 activated the expression of NCED2, which was inferred from the increased promoter activity, as indicated by the enhanced LUC/REN ( Figure 5F). These results clearly demonstrate that MYC2 positively regulates the transcription of NCED2 and ABA accumulation in tomato cold stress.

Discussion
As a major abiotic stress, low temperature inhibits plant growth and causes considerable losses of crop yields. Tomato is a warm-climate species and cold stress adversely affects its growth and development at nearly all stages, including vegetative, reproductive, and fruiting. It is, thus, critical to understand how tomato responds to cold stress at the physio- logical and molecular levels. Initially identified as a defense hormone against pathogen and insect attack, JAs have recently emerged as crucial players in adaptation to cold stress in plants. The positive role of JAs in cold tolerance has been observed in a variety of species, such as Arabidopsis [33], banana [36], rice [51], trifoliate orange [39] and tomato [35]. Several mechanisms for JA-mediated cold tolerance have been proposed in these studies; however, our knowledge on the mechanisms by which JA regulates cold response is still rudimentary, in particular in tomato. ABA is a well-known phytohormone that is vital for a plethora of abiotic stress responses, including cold response [52,53]. Previous studies have established the crosstalk between JA and ABA during stress responses [42,54]; however, whether and how these two hormones interact to regulate tomato cold response are still unclear. In this work, we determined that JA promotes tomato cold tolerance by upregulating the ABA biosynthesis gene NCED2. We showed that NCED2 was responsive to both cold stress and JA and silencing NCED2 resulted in decreased ABA accumulation and increased cold sensitivity in tomato. Furthermore, blocking ABA biosynthesis attenuated JA-induced cold tolerance. We also revealed that the JA-triggered expression of NCED2 partially relied on MYC2, which is a core regulator of the JA signaling pathway.
Previous reports have pointed to the importance of JA in the tomato cold response. In this study, we observed that JA accumulation was enhanced under cold stress, substantiating the involvement of JA in the tomato cold response. Intriguingly, JA appears to be related with ABA in response to cold conditions in tomato, as the accumulation of JA was concurrent with that of ABA, with both peaking at 12 h following cold treatment. The association of JA with ABA in the tomato cold response was further supported by the applications of exogenous MeJA and DIECA, a biosynthesis inhibitor of endogenous JA, under cold conditions. Our data herein showed that whereas MeJA promoted cold-triggered ABA accumulation, DIECA attenuated cold-induced ABA accumulation in tomato, implying the crosstalk between JA and ABA. Similar relations between JA and ABA were also observed in drought-stressed Arabidopsis plants [55]. However, contrary to what we observed in this study, a previous work reported that a decreased level of JA did not exert a pronounced effect on the accumulation of ABA in tomato under drought conditions [56]. Together, these results suggest the complexity of the crosstalk between JA and ABA, which may be determined by the type of stresses and species.
Our observation that JA promoted ABA accumulation raises an interesting question of how JA acts in the biosynthesis of ABA under cold stress. It has been established that NCED functions as a rate-limiting enzyme during ABA biosynthesis [43,57]. The genes that encode NCED usually form a multigene family. In tomato, three NCED genes, including NCED1, NCED2, and NCED6, have been identified to play major roles in ABA biosynthesis [58,59]. We, thus, hypothesized that NCED might be targeted by JA. To validate our hypothesis, we performed transcriptome analysis in an attempt to identify JA-targeted NCED genes. NCED2 was identified to be upregulated by JA. The regulation of NCED2 transcription by JA was further corroborated by our time-course analysis of NCED2 expression upon MeJA treatment, in which the transcript abundance of NCED2 was remarkably elevated. It is worth nothing that a previous study demonstrated that JA deficiency led to increased NCED1 transcript accumulation in tomato in response to water stress [56]. Interestingly, another report shows that JA induced the expression of NCED3 to regulate stomatal closure [60]. Thus, our results and those of previous studies highlight that JA is crucial for the regulation of ABA biosynthesis genes in response to environmental stress.
NCED genes have been previously shown to be responsive to salinity, drought and cold. After establishing that JA positively regulates the expression of NCED2, we next sought to understand the role of JA-NCED2 in tomato under cold conditions. Our timecourse examination of NCED2 expression in tomato leaves under cold stress revealed that NCED2 was responsive to cold stress and was rapidly induced, implying that NCED2 may be involved in the tomato cold response. Here, we found that the silencing of NCED2 via VIGS led to enhanced cold sensitivity in tomato, as evidenced by the decreased Fv/Fm and increased REL levels. The altered cold sensitivity may be associated with the change in the level of endogenous ABA, as ABA accumulation was markedly reduced in NCED2 VIGS plants compared with that in control plants under cold stress. Furthermore, exogenous MeJA significantly promoted cold tolerance, while exogenous fluridone (Flu), an ABA biosynthesis inhibitor, abated the effects of MeJA on cold tolerance.
As a core component of the JA signaling pathway, MYC2 has been verified to play essential roles in multiple developmental processes and stress responses. Whether MYC2 is involved in JA-induced NCED2 expression and ABA accumulation under cold stress remains unanswered. In the current work, multiple lines of evidence support the role of MYC2 in regulating NCED2 expression, ABA accumulation, and cold tolerance in tomato. First, the suppressed expression of MYC2 in MYC2-RNAi plants led a reduction in NCED2 in tomato under cold stress. Second, two potential MYC2 binding sites were identified in the 2 kb promoter region of NCED2. Third, dual-luciferase assays demonstrated that MYC2 activated the transcription of NCED2. Lastly, in accordance with decreased NCED2 expression, ABA accumulation was largely reduced in MYC2-RNAi tomato plants under cold stress. Consistently, MYC2-RNAi tomato plants also demonstrated decreased cold tolerance, as indicated by the increased REL in tomato leaves following cold treatment. Hence, MYC2 acts as a crucial regulator in JA-triggered ABA accumulation and cold resistance in tomato.

Plant Materials and Growth Conditions
Three genotypes of tomato (Solanum lycopersicum 'MicroTom'), including wild-type tomato, transgenic tomato (MYC2-RNAi) and VIGS tomato (TRV-NCED2), were used as plant materials in this work. Tomato seedlings were grown in substrate that contained peat and vermiculite (2/1, v/v) in a growth chamber, with the following growth conditions: 14 h light/8 h dark cycles with the light density of 200 µmol m −2 s −1 , 26 • C/21 • C (light/dark), and ∼70% relative humidity.

Cold Treatments
For the cold treatments, tomato plants were exposed to 4 • C at the 4-leaf stage. To determine the JA and ABA accumulations, cold stress was imposed on the tomato plants for 24 h. To determine the relative expression of NCED2 in response to MeJA, young leaves were detached and subjected to MeJA treatment by floating on 50 µM of MeJA solution in petri dishes for 24 h. To determine the relative expression of NCED2 in response to cold stress, tomato seedlings were subjected to cold stress for 24 h and leaf samples were collected at 0, 3, 6, 12 and 24 h following cold treatment. To understand the importance of JA in NCED2 expression and ABA accumulation under cold stress, we first sprayed tomato plants at the 4-leaf stage with mock solution, MeJA (100 µM), or DIECA (2 mM), and 12 h later, these plants were subjected to cold stress. To understand how MYC2 might regulate the expression of NCED2, MYC2-RNAi tomato plants at the 4-leaf stage were grown under cold conditions for 24 h.

Assessment of Cold Tolerance
Two cold-sensitive parameters, Fv/Fm and REL (relative electrolyte leakage), were measured to determine the cold tolerance of tomatoes. Fv/Fm measurements were performed following the procedures reported previously [8]. After cold treatment, to obtain the minimal fluorescence (Fo), we placed tomato plants in the dark for 30 min and following dark adaptation, Fo was measured with a portable chlorophyll fluorometer. After completing measurements of Fo, we applied a saturating pulse to the leaves to obtain the maximal fluorescence (Fm).
REL was measured as previously reported [38]. In brief, after cold treatment, tomato leaves were detached and incubated in deionized water to obtain the initial conductivity (C1). Leave materials were subsequently boiled to obtain the maximum conductivity (C2). REL was expressed as C1/C2 × 100%.

Expression Analysis of NCED2
qRT-PCR was performed to quantify the relative transcript abundance of NCED2. Briefly, we first isolated the total RNA using an RNAprep Pure Plant Kit (TIANGEN, Beijing, China). Then, we synthesized cDNA using the total RNA as a template. PCR reactions were carried out with a commercial kit (Premix Ex Taq, TaKaRa Bio, Dalian, China). ACTIN2 in tomato was used as an internal control. All measurements were performed with two technical and three biological replicates. The primers for relative gene expression analysis of NCED2 were GAGGACGATGGATATATTCTTGCA (forward) and AGTCCTTTGAGCTAATGAATGTCC (reverse). The primers of ACTIN2 used for qRT-PCR analysis were TCCCAGCAGCATGAAGATTAAG (forward) and CCTGTGGACAATG-GATGGAC (reverse).

Quantification of JA and ABA
Accumulations of JA and ABA in tomato leaves after cold treatments were measured according to a previous study [28]. To extract JA and ABA, we first ground the leaves to a fine powder with liquid N 2 , which was then homogenized in ethyl acetate. Ethyl acetate was spiked with D6-ABA and D5-JA, which acted as internal standards. The homogenate was subsequently centrifuged at 18,000× g for 10 min at 4 • C. Then, the supernatant was collected and evaporated to dryness under nitrogen gas. We resuspended the pellets in methanol (70%, v/v) and centrifuged them at 18,000× g for 2 min. The supernatants were collected and subjected to HPLC-mass spectrometry analysis. Endogenous JA and ABA levels were expressed as ng/g fresh leaves.

Virus-Induced Gene Silencing (VIGS) of NCED2
To knock down the expression of NCED2, we employed virus-induced gene silencing using tobacco rattle virus (TRV) vectors, TRV1 and TRV2, according to a previous study [12]. A fragment (300 bp) of NCED2 was selected using the SGN VIGS Tool. The fragment was then amplified and ligated into the TRV2 vector, and an empty TRV2 vector was used as a control. We then introduced all the constructs into Agrobacterium tumefaciens GV3101. Cultures of Agrobacterium tumefaciens with TRV1, TRV2-NCED2, or TRV2 were mixed and the mixture was used to inoculate fully expanded cotyledons. The infected seedlings were maintained in a growth chamber for four weeks, and then leaves were collected for qRT-PCR analysis of NCED2 expression. Plants with~80% reductions in NCED2 expression were selected for further experiments.

Dual-Luciferase Assays
We performed dual-luciferase assays using tobacco (Nicotiana benthamiana) leaves, as described in a previous study [40]. Two vectors, including pGreen II 0029 62-SK (SK) and pGreen II 0800-LUC (LUC), were used for the dual-luciferase assays. We first amplified the full-length coding fragment of MYC2 and ligated it to the pGreen II 0029 62-SK (SK) vector to produce the effector construct. Then, we cloned the promoter fragment (2 kb) of NCED2 and ligated it into the pGreen II 0800-LUC (LUC) vector to generate the reporter construct. Both constructs were then introduced into the Agrobacterium tumefaciens strain GV3101. We then mixed cultures of Agrobacterium tumefaciens that harbored the effector construct or the reporter construct and used the mixture to inoculate tobacco leaves. A Dual-Luciferase Assay Kit (Promega, Madison, WI, USA) was used to measure the LUC and REN activities three days after inoculation, and the ratio of LUC/REN was calculated.

Conclusions
In summary, we demonstrate the crosstalk between JA and ABA at the biosynthetic level in tomato leaves under cold stress. JA triggers the expression of the ABA biosyn-thesis gene NCED2 through MYC2, eventually promoting ABA accumulation and cold resistance in tomato. Finally, we propose a working model of JA-mediated cold tolerance via ABA biosynthesis ( Figure 6). This work expands our understanding of JA-mediated cold tolerance and the crosstalk between JA and ABA in plants under stress conditions.

Conflicts of Interest:
The authors declare no conflict of interest.

Conflicts of Interest:
The authors declare no conflict of interest.