1. Introduction
Terpenoids are the most common and diverse class of secondary metabolites in plant volatiles; they can be produced by almost all plant organs, including roots, stems, leaves, flowers, fruits, and seeds [1], and they play important roles in the growth and development of plants themselves. Terpenoids have isoprene as their structural unit and mainly include monoterpenes, sesquiterpenes, diterpenes, and triterpenes [2,3]; monoterpenes and sesquiterpene compounds are the main volatile substance components produced by plants. Terpenoids can act as signaling molecules to mediate plant defense responses to phytophagous insect feeding and play an important role in the resistance to pathogenic microbial attacks, among other roles [4,5].
There are two pathways that are important for the synthesis of plant terpenoids: the mevalonate (MVA) pathway located in the cytoplasm and the methylerythritol-4-phosphate (MEP) pathway located in the plastid [6]. 1-deoxy-D-xylulose-5-phosphate synthase (DXS) is the first key enzyme of the MEP pathway that converts pyruvate and 3-phosphoglycerol aldehyde to produce the first key intermediate, 1-deoxy-xylulose-5-phosphate (DXP). This is considered to be the rate-limiting step in the synthesis of isopentenyl pyrophosphate (IPP) and dimethylpropenyl pyrophosphate (DMAPP) via the MEP pathway [7,8]. The overexpression of the DXS gene in Arabidopsis leads to a significant increase in the terpene content [9,10]. The Ginkgo DXS gene is expressed in all trophic organs, and it is induced and regulated by methyl jasmonate, whereas the synthesis of ginkgolides is positively correlated with DXS expression [11]. These observations confirm that the DXS gene is an important regulatory site in the terpene metabolic pathway and can effectively influence the accumulation of secondary metabolites in plants.
Tomatoes (Solanum lycopersicum L.), belonging to the tomato genus of the Solanaceae family, are an important vegetable crop around the world, widely cultivated in the north and south of China. Tomatoes are rich in soluble sugar, organic acids, vitamins, and other nutrients, and consumers love their rich flavor. Tomatoes can be infested by phytophagous insects during growth, resulting in a decrease in the yield and quality of tomatoes [12]. Plants stimulate their own defense mechanisms when attacked by phytophagous insects. For example, plants can produce physical barriers and secondary metabolites, and they can induce the expression of relevant genes for direct defense [13,14]. Flavonoids are important secondary metabolites and are phenolic compounds. They are capable of hindering the feeding of pests and of affecting their growth, development, and reproduction [15,16]. Additionally, plants release volatile organic compounds as an indirect defense after an infestation to attract natural enemies to feed on or parasitize the pest [17]. Using the plants’ own defense mechanisms to control pests is an environmentally friendly approach. It has been found that all tissue parts of a tomato are rich in terpenoids. Terpenoids are not only able to participate in the plant’s defense response, acting directly or indirectly against phytophagous insects, but they can also act against disease infiltration [18,19]. The expression of the tomato DXS gene and the regulation of the synthesis of volatile terpenoids may influence a tomato’s defense response to adversity.
The DXS gene encodes the first key enzyme in the terpenoid MEP pathway in tomatoes. In this study, we identified the DXS family members and bioinformatically analyzed three SlDXS gene-encoded proteins. We also explored the effects of different abiotic stresses on the expression of the tomato DXS gene. A virus-induced gene silencing (VIGS) vector was constructed for the silencing of a SlDXS gene, and the content of photosynthetic pigments in the silenced plants was analyzed. Real-time fluorescence quantification PCR (RT-qPCR) was used to analyze its induction of exogenous MeJA on silenced plants. This study provides a basis for an in-depth investigation of terpenoid metabolic pathways and molecular regulatory mechanisms in tomatoes in addition to a theoretical basis for further research on tomatoes’ resistance to external stress.
2. Materials and Methods
2.1. Identification of the DXS Gene Family in Tomatoes
The tomato genome-wide data files and genome annotation files (ITAG4.0) were downloaded from the tomato genome website (
2.2. Physicochemical Analysis of Proteins of the DXS Gene Family in Tomatoes and Prediction of Their Subcellular Localization
The protein physicochemical properties of SlDXS were analyzed using the online website expasy (
2.3. Prediction of Secondary and Tertiary Structures of Tomato DXS Gene Family Members
The DXS protein’s secondary structure was predicted using Prabi (
2.4. Phylogenetic Analysis of the Tomato DXS Gene
MEGA7 software was used to conduct a multiple sequence comparison of the DXS gene families of 13 species, including Solanum lycopersicum, Arabidopsis thaliana, Zea mays, Oryza sativa, Populus trichocarpa, Capsella rubella, Ricinus communis, Medicago truncatula, Nicotiana tabacum, Ginkgo biloba, Hevea brasiliensis, Salvia miltiorrhiza, and Pinus densiflora, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method. The parameters were set as the Poisson correction, pairwise deletion, and bootstrap test (1000 repetitions), and the phylogenetic tree was beautified using the iTOL webpage (
2.5. Gene Structure and Conserved Motif Analysis of DXS Gene Family in Tomatoes
The gene structure information of the tomato DXS gene family members was analyzed using the Tbtools software v1.098775 [23], and the SlDXS gene structure was analyzed using the NCBI’s online website (
2.6. Analysis of Promoter Cis-Acting Elements of the DXS Gene Family in Tomatoes
The 2000 bp sequences upstream of the transcription start site of the SlDXS gene were extracted using TBtools, and the cis-acting elements in the promoter regions were predicted using PlantCARE (
2.7. Abiotic Stress Treatment of Plant Materials
A ‘Micro Tom’ tomato was used as the material for stress treatment. The tomatoes were subjected to abiotic stress until they reached the six-leaf stage under normal conditions (light/dark for 16 h/8 h, 25 °C/20 °C, photosynthetic photon flux density of 200 µmol·m−2·s−1). The tomato leaves were sprayed with concentrations of 100 μmol/L of methyl jasmonate, gibberellin, and abscisic acid and 100 mmol/L of NaCl. The fifth and sixth true leaves at 0 h, 1 h, 3 h, 6 h, 12 h, 24 h, and 48 h were taken for quantitative analysis. After liquid nitrogen quick-freezing, the leaves were stored in the refrigerator at −80 °C, and three biological replicates were performed.
2.8. Expression Analysis of the DXS Gene in Tomatoes
The total RNA of tomato was extracted using the Vazyme FastPure® Plant Total RNA Isolation Kit (
2.9. Cloning the SlDXS Gene and Constructing the VIGS Silencing Vector
SnapGene was used to design primers specific to the SlDXSs gene (Supplementary Tables S2 and S4), and tomato cDNA was used as a template to amplify the target gene. The PCR program was as follows: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 15 s, annealing at 59 °C for 15 s, and extension at 72 °C for 30 s, for a total of 34 cycles.
The KpnI and EcoRI restriction endonucleases were selected to double digest the pTRV2 vector plasmid, and the SlDXS fragment was inserted into the pTRV2 silencing vector. The pTRV2-SlDXS recombinant vector was verified through digestion. pTRV1 and the pTRV2-SlDXS recombinant vector were transformed into Agrobacterium GV3101. The single colonies containing pTRV1 and recombinant vector pTRV2-SlDXS were picked and inoculated into 400 μL of LB liquid medium (Kan 100 mg/L, Rif 50 mg/L), and then incubated for 6 h at 28 °C at 200 rpm. An amount of 200 μL of the above bacterial solution was added into 10 mL of LB liquid medium (Kan 100 mg/L, Rif 50 mg/L), and then incubated for 12 h at 28 °C at 200 rpm. The bacterial cells were collected by centrifugation at 8000 rpm for 10 min, the bacteria were resuspended in a VIGS infiltration solution (10 mmol/L of MgCl2; 10 mmol/L of MES; 200 μmol/L of AS), and the OD600 was adjusted to about 0.8. The cells were allowed to stand at room temperature for 4 h, and the resuspension of pTRV1 with pTRV2-SlDXS was mixed at a volume ratio of 1:1 and injected into tomato cotyledons. The inoculum was aspirated with a 1 mL syringe and gently injected into the dorsal surface of the plants’ cotyledons, which were protected from light for 24 h. After that, the plants were transferred to a light incubator for further incubation.
2.10. Treatment of Silencing Plant Materials
A ‘Micro Tom’ tomato was used as the material for the silencing treatment, and the infested plants were cultured in a light incubator (photoperiod: 16 h/8 h light/dark cycle; temperature: 25 °C/20 °C day/night; PPFD: 200 µmol·m−2·s−1) for three weeks. RT-PCR was performed on the silenced plants to validate the transcription of TRV2 in order to obtain the positive plants. The expression level of the SlDXS2 gene was measured using Actin as an internal reference gene, and the relative expression level of the gene was calculated using the 2−ΔΔct method. The content of photosynthetic pigments in tomato leaves was determined by the ethanol extraction colorimetric method [25]. The leaves of the positive plants were treated with MeJA, and the samples were collected 6 h later. Flavonoids were determined using a kit from Comin Biotechnology (Suzhou, China). Three biological replicates were set up.
3. Results
3.1. Identification and Physicochemical Analysis of the DXS Gene Family in Tomatoes
Three members of the DXS gene family in tomatoes were identified using bioinformatics methods, and according to the existing nomenclature of Arabidopsis and phylogenetic developmental analyses, they were sequentially named SlDXS1, SlDXS2, and SlDXS3. Protein characterization revealed (Table 1) that the amino acid lengths of the proteins encoded by the family members were 719, 714, and 709aa, and the molecular weights ranged from 77,605.63 to 77,172.8 Da. The theoretical pI values of the SlDXS family members were all less than 7, and all of them were acidic proteins. SlDXS proteins do not have a signal peptide for any of the amino acids and are non-secretory proteins, and both SlDXS1 and SlDXS2 are hydrophilic, with a hydrophobicity of less than 0. The instability index was greater than 40, which made them unstable acidic proteins. An analysis of the subcellular localization of the proteins showed that SlDXS proteins are all predicted to be localized in the chloroplasts [26].
3.2. Secondary and Tertiary Structure Analyses of the DXS Gene Family in Tomatoes
A predictive analysis of the secondary structures of tomato DXS proteins revealed (Table 2) that all three proteins consisted of α-helices, extended strands, β-turns, and random coiling in their secondary structures. The number of amino acid residues accounted for was dominated by α-helices and random coils, followed by extended chains, and β-turns. Further analysis of the tertiary structure (Figure 1) showed that the templates for SlDXS1, SlDXS2, and SlDXS3 proteins were all 7bzx.1.A, and the sequence identities were 88.75%, 73.93%, and 58.24%, respectively.
3.3. Phylogenetic Analysis of the DXS Gene Family in Tomatoes
The SlDXS proteins were combined with the sequences of proteins encoded by the DXS genes of 12 species, including Arabidopsis thaliana, Oryza sativa, Zea mays, Nicotiana tabacum, Salvia miltiorrhiza, and Ricinus communis, to perform phylogenetic analyses and construct a phylogenetic tree using the neighbor-joining (NJ) method (Figure 2). The results of the phylogenetic analysis showed that the DXS gene family members in all plants co-clustered into three branches, and the three tomato DXS genes were located in different groups. The DXS I branch had the largest number of members, including AtDXS1 and AtDXS2 in Arabidopsis, and SlDXS1 was more closely related to NtDXS1 and SmDXS1. SlDXS1 and NtDXS2 clustered into one branch, suggesting a high degree of affinity. There were fewer members in the DXS III branch, including species such as Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Zea mays, Ricinus communis, and Capsella bursa-pastoris, whereas the tomato SlDXS3 was closer to AtDXS3. Therefore, it was hypothesized that the SlDXS1, SlDXS2, and SlDXS3 genes may have different functions.
3.4. Analysis of the Gene Structure and Conserved Motifs of the DXS Gene Family in Tomatoes
In order to further analyze the structural characteristics of the SlDXS gene family, three genes of the tomato DXS gene family were subjected to relevant intron and exon analyses (Figure 3A), and the results showed that the SlDXS1, SlDXS2, and SlDXS3 genes all have 10 exons and 9 introns. The tomato DXS protein’s conserved motifs were analyzed using the online MEME software, and eight motifs were used to analyze the gene sequences (Supplementary Table S3). There were eight motifs for all three genes, and the ranking order was fixed (Figure 3B). This indicates that there are highly conserved motifs in the DXS protein family and that the DXS gene family is highly conserved. A predictive analysis of the structural and functional domains of the tomato DXS proteins revealed that all three proteins had DXS structural domains (Figure 3C), indicating that the SlDXS1, SlDXS2, and SlDXS3 proteins all belong to the DXS superfamily.
3.5. Analysis of Cis-Acting Elements in Promoters of the DXS Gene Family in Tomatoes
The promoter sequences of the first 2000 bp of the tomato SlDXS genes were obtained using the TBtools software, and the promoter cis-acting elements of the SlDXS genes were analyzed using the PlantCARE online website. The results showed that the promoter regions of the SlDXS gene family contain a variety of cis-acting elements with different roles, indicating that the three SlDXS genes may be involved in different physiological and metabolic regulatory pathways. A total of 121 cis-acting elements were detected in the 2000 bp region of the upstream promoter of the tomato SlDXS gene family. They were classified into four categories: hormone-responsive elements, light-responsive elements, growth and development elements, and stress-related cis-acting elements (Figure 4). The results showed that there were 6 hormone-related regulatory elements, including 4 methyl jasmonate regulatory elements (CGTCA-motif and TGACG-motif), 10 ABA-responsive regulatory elements (ABRE), 2 salicylic acid-responsive regulatory elements (TCA-element), 1 growth hormone-responsive element (TGA-element), and 19 gibberellin-responsive regulatory elements (ERE). There were two elements related to growth and development, including meristematic tissue expression (CAT-box) and endosperm expression (AACA-motif). A total of 43 stress-related response regulatory elements were found, including MYC, ARE, and W-box. There were 11 different elements related to light response regulation, such as the 1-box, GA-motif, and ATCT-motif. Light-responsive elements were present in each gene family member, suggesting that expression of the SlDXS gene may be induced or repressed by light. The SlDXS gene family members contain hormone-responsive elements, and it is hypothesized that tomato SlDXS genes play an important role in hormone responses. The SlDXS2 gene contains two methyl jasmonate regulatory elements (TGACG-motif), which presumably respond to MeJA expression.
3.6. Analysis of the Induced Expression Pattern of the Tomato DXS Gene Family
To examine the expression levels of the SlDXS gene family members, the tomato leaves were treated with MeJA, GA, ABA, and salt stress factors, respectively. The results showed that, after the tomato leaves were treated with MeJA, the expression levels of SlDXS1 and SlDXS3 showed decreasing trends, which were minimized at 24 h and 6 h, respectively. The expression level of SlDXS2 increased at 3 h and 24 h, and it peaked at 3 h (Figure 5A). This indicates that MeJA was able to induce the expression of the SlDXS2 gene and significantly increase its expression level. After the GA treatment was applied, the expression of SlDXS2 increased from 1 h to 9 h, and it peaked at 1 h; its expression was 4.22 times higher than that of the control group. The expression of SlDXS3 increased rapidly at 1 h and peaked at 1 h, and its expression was more than 2.23 times higher than that of the control group. The expression of SlDXS1 showed a decreasing tendency (Figure 5B). This indicates that GA induced the expression of SlDXS2 and SlDXS3, while SlDXS1 was not responsive to GA. After the tomato leaves were treated with ABA, the expression levels of both SlDXS1 and SlDXS2 showed decreasing trends, which were minimized at 12 h and 1 h. The expression of SlDXS3 increased at 1 h and 9 h, and its expression levels were 1.55 and 1.51 times higher than that of the control, respectively (Figure 5C). This indicates that SlDXS3 responded slower to ABA. Under different hormone treatments, SlDXS1, SlDXS2, and SlDXS3 had different responses and presumably different functions. After the salt stress treatment was applied, the expression of SlDXS1 increased transiently at 1 h and decreased from 3 h to 48 h. The expression of SlDXS2 increased at 1 h, 6–12 h, and 48 h; peaked at 6 h; and was 7.64 times higher than that of the control group. The expression of SlDXS3 increased at 3 h and 48 h and peaked at 48 h (Figure 5D). The expression levels of SlDXS1 and SlDXS2 increased rapidly at 1 h, indicating that they had more rapid responses to NaCl.
3.7. Cloning the SlDXS Genes and Constructing the VIGS Silencing Vector in Tomatoes
To further investigate the biological function of SlDXS genes, VIGS technology was utilized to silence SlDXS genes in tomatoes. Fourteen days after the Agrobacterium infection of tomato cotyledons, photobleaching appeared on the leaves of tomato seedlings injected with the pTRV2-SlDXS1 infection solution (Figure 6B). One month after inoculation, photobleaching appeared in most leaves of the plants infected with pTRV2-SlDXS1 (Figure 6F). In contrast, the albino phenotype was not presented in wild-type plants and the plants injected with pTRV2-SlDXS2 and pTRV2-SlDXS3 infection solutions (Figure 6F–H).
The expression levels of the SlDXS genes in plants that underwent different treatments were detected through an RT-qPCR using Actin as an internal reference gene, and the results showed that pTRV2-SlDXS resulted in a significant decrease in the expression of the SlDXS genes compared to the control group (Figure 6b).
The content of photosynthetic pigments was determined in wild-type and silent plants. As shown in Figure 6c–e, the content of photosynthetic pigments was reduced in all silenced plants compared to the wild type. Silenced plants injected with pTRV2-SlDXS1 showed significant reductions in chlorophyll a, chlorophyll b, and carotenoids by 46.63%, 38.99%, and 46.02%, respectively. Silenced plants injected with the pTRV2-SlDXS3 infection solution showed a reduction of 16.14% and 15.84% in chlorophyll a and chlorophyll b, respectively, whereas there was no significant difference in carotenoid content. There was no significant difference in the photosynthetic pigment content of tomato plants silencing the SlDXS2 gene. It was shown that the SlDXS1 gene is associated with the synthesis of chlorophyll and carotenoids, and the silencing of the SlDXS1 gene can cause a decrease in the content of photosynthetic pigments in tomatoes.
3.8. Analysis of TRV2-SlDXS2 Gene Response to MeJA
As shown in Figure 5A, MeJA increased the expression of the tomato SlDXS2 gene. The following procedures were conducted in order to further investigate the response of SlDXS2 to MeJA and its biological function.
The tomato leaves were treated with MeJA, and the samples were collected after 6 h for a gene expression analysis and the determination of physiological indices. The tomato SlDXS2 gene was analyzed using an RT-qPCR after MeJA treatment. The results showed that the expression of the SlDXS2 gene was increased in both the wild-type and silenced plants, but MeJA promoted the expression of the SlDXS2 gene at a lower level in the silenced plants compared with the wild-type plants (Figure 7A). This indicates that MeJA reduced the induction of SlDXS2 in the silenced plants.
Flavonoids are important secondary metabolites that help plants resist pests and diseases, whereas terpenoids have a role in helping plants avoid pests [27,28]. The flavonoid content was determined after the MeJA treatment was applied to silenced and wild-type tomato plants, and the results showed that the flavonoid contents of both the wild-type and silenced plants were elevated after the MeJA treatment; however, compared with the wild-type plants, the MeJA-induced synthesis of flavonoids was lower in the silenced plants (Figure 7B). This suggests that MeJA’s promotion of secondary metabolism in tomatoes was reduced after SlDXS2 silencing.
The physiological indicators and the results of the gene expression analyses indicated that MeJA was able to significantly induce the expression of the tomato SlDXS2 gene and promote the synthesis of secondary metabolites; they also indicated that the SlDXS2 gene was capable of responding to MeJA in silenced plants, but at a lower level of induction relative to that of wild-type plants.
4. Discussion
Terpenoids play important roles as chemical signaling substances during indirect defense responses, such as by helping plants avoid pests and natural enemies, and they are involved in plant-to-plant and plant–insect interactions [27]. The DXS enzyme is the first key enzyme in the MEP pathway, which is one of the terpene synthesis pathways, and the DXS gene is also the rate-limiting enzyme gene in the MEP synthesis pathway [29]. It was shown that the tissue expression pattern of the DXS gene in C. blini was positively correlated with the tissue accumulation pattern of the diterpene substance artemisinin, suggesting that the overexpression of the DXS gene may increase the synthesis of artemisinin in C. blini [30]. The overexpression of the DXS2 gene in the hairy roots of S. miltiorrhiza was able to significantly promote the accumulation of tanshinones [31]. It was also demonstrated that the DXS gene is important for terpenoid synthesis.
DXS is currently confirmed to be a small gene family in several species, such as A. thaliana [32], Z. mays [33], and A. annua [34], usually containing between two and four members. In this study based on the tomato genome, three tomato DXS genes were identified, which is the same number of DXS gene family members as in A. thaliana, O. sativa, and Z. mays, and DXS gene family members have similar gene structures and conserved motifs in many species. It was found that AtDXS3 is not involved in primary and secondary metabolism [35]. It was found that the SlDXS3 protein does not have the same conserved residues as SlDXS1 and SlDXS2. Therefore, the SlDXS3 protein may not be able to bind to TPP cofactors [26]. An analysis of the subcellular localization of the proteins showed that SlDXS proteins are all predicted to be localized in the chloroplasts. This is consistent with the fact that the three DXS genes of Morus notabilis are located in chloroplasts [36]. The MEP pathway occurs in plastids, so it is reasonable that the SlDXS genes are located in chloroplasts [36]. It was found that the DXS gene family contains three subfamilies, namely DXS I, DXS II, and DXS III, each with different functions [33]. Specifically, type I DXS genes are mostly housekeeping genes and may be involved in plants’ primary metabolism, type II DXS genes mostly encode for proteins that participate in plants’ secondary metabolism, and type III genes may be involved in the biosynthesis of related substances on which the survival of plants depends [37]. Through a phylogenetic analysis of tomatoes with A. thaliana, Z. mays, and O. sativa species, the SlDXS genes were divided into three different branches, and it was hypothesized that DXS enzymes have different functions. SlDXS1 was in the same branch as the AtDXS1 and SmDXS1 species, which indicated that SlDXS1 might play the role of housekeeping genes [38]. Additionally, SlDXS2 was in the same branch as the SmDXS2 and MtDXS2 species [39], and SlDXS3 is a type III gene that may be involved in the synthesis of MEP pathway derivatives, which is consistent with the function of AaDXS4 in artemisinin.
Photosynthetic pigments are important substances for primary production in plants and their levels are related to plant growth and development [40]. When subjected to adversity stress, the chlorophyll content decreases [41]. The CrDXS1 gene in Citrus reticulata is positively correlated with the accumulation of carotenoid content [42]. The overexpression of the AtDXS1 gene from Arabidopsis thaliana increased the chlorophyll and carotenoid content [43]. The overexpression of the GmDXS gene of Glycine max significantly increased the photosynthetic pigment content [44]. Silencing the SlDXS genes in tomato leaves using VIGS technology showed that silencing the SlDXS1 gene resulted in the photobleaching of plant leaves. Meanwhile, silencing SlDXS2 and SlDXS3 did not show a bleaching phenotype. This indicates that the SlDXS1 gene is involved in chlorophyll synthesis.
The results of the prediction and analysis of the tomato DXS gene family promoter’s cis-acting elements showed that DXS may respond to a variety of abiotic stresses and hormones. After the salt stress treatment, SlDXS1, SlDXS2, and SlDXS3 peaked in expression at different times, suggesting that the expression of the tomato DXS gene can be increased by salt stress. The expression levels of both the AaDXS2 and AaDXS3 genes were significantly increased in Artemisia annua after salt stress treatment [34]. Populus trichocarpa seedlings showed an increased expression of the PtDXS gene after NaCl treatment [45]. The PmDXS gene showed an upward trend after salt stress in Pinus massoniana [46]. Under the treatment of different exogenous hormones, the expression of the tomato SlDXS genes varied and showed different patterns, which may be related to the different functions of the SlDXS gene family. After the tomato leaves were treated with ABA, the expression levels of both SlDXS1 and SlDXS2 showed decreasing trends, whereas SlDXS3 was responsive to ABA. After the GA treatment, the expression levels of SlDXS2 and SlDXS3 increased rapidly, indicating that they had more rapid responses to GA. After the exogenous GA treatment of Camellia sinensis, the expression of the CsDXS gene reached its maximum at 4 h, which was 1.3 times of that of CK [47]. In the process of regulating a plant’s metabolism, different stress conditions can have inducing, promoting, or inhibiting effects, thus affecting the formation and accumulation of secondary metabolites in plants.
It was found that exogenous MeJA could significantly regulate the accumulation of secondary metabolites, and exogenous MeJA induced the accumulation of volatile monoterpenes in grape pericarp [48]. WRKY transcription factors in P. grandiflorus can regulate the synthesis of triterpenoid compounds in response to MeJA [49]. Exogenous MeJA treatment significantly increases lavender’s monoterpene and sesquiterpene contents [50]. Applying exogenous MeJA treatment to Goosegrass rhizomes leads to an increase in the triterpenoid saponin content [51]. It was found that the expression of MnDXS2A and MnDXS2B is up-regulated by the exogenous MeJA treatment of mulberry seedling leaves, which was hypothesized to be possibly related to certain metabolites in the plant and plant defense system [36]. In this study, where MeJA was used to treat tomato leaves, SlDXS1 and SlDXS3 were not responsive to MeJA, whereas the expression of SlDXS2 was up-regulated to the maximum at 3 h, indicating that SlDXS2 can be significantly induced by exogenous MeJA. This may be related to the presence of a cis-acting element (TGACG-motif) in the tomato SlDXS2 gene in response to methyl jasmonate, which may explain why the MeJA treatment significantly induced the expression of the key SlDXS2 gene in tomatoes.
As a common signaling substance in plants, methyl jasmonate (MeJA) induces the production of volatiles to help plants avoid pests, thus reducing the damage caused to plants [52]. The silencing of the SlDXS2 gene in tomato leaves using VIGS technology showed that SlDXS2 was able to respond to an exogenous MeJA expression in silenced plants. However, the induction level was low. It was found that SlDXS2 was trauma-responsive in RNAi plants, but the expression level was significantly reduced [53]. Flavonoids are important secondary metabolites synthesized by plants, that help them to develop insect resistance. After plants are infested by insect pests, flavonoids are synthesized and accumulated in a plant’s body, and the higher their content, the higher the plant’s ability to resist insects [28]. The flavonoid content of tomato leaves increased significantly after their treatment with MeJA. However, MeJA induced the synthesis of flavonoids at lower levels in the silenced plants compared to the wild-type plants. This indicates that MeJA promotes secondary metabolism in tomato plants less after SlDXS2 silencing. This study lays the foundation for future studies.
5. Conclusions
In this study, three members of the tomato DXS gene family were identified, and their physicochemical properties, gene structures, phylogenetic relationships, and cis-acting elements were analyzed using bioinformatics. An RT-qPCR was used to analyze the expression patterns of the DXS genes under different stresses, and it was found that the expression of the SlDXS2 gene was increased by an exogenous MeJA treatment. The silencing of the SlDXS gene verified that SlDXS1 is associated with the accumulation of photosynthetic pigments through a virus-induced gene silencing (VIGS) method study. The silencing gene known as pTRV-SlDXS2 was also verified to be capable of responding to an exogenous MeJA expression. This study provides a reference for the further elucidation of the biological function of SlDXS genes from the terpenoid synthesis pathway in tomato plants.
Conceptualization, F.Z. and M.H. (Maomao Hou); data curation, H.G., M.H. (Mingxuan Han), J.L. and L.L.; investigation, J.T., S.L. and H.Z.; writing—original draft preparation, H.G.; writing—review and editing, H.G. and J.L.; project administration, F.Z. All authors have read and agreed to the published version of the manuscript.
Data are contained within the article and
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 2. Phylogenetic tree analysis of tomato SlDXS1, SlDXS2, and SlDXS3 proteins and DXS proteins from other species. The SlDXS proteins are marked with red stars.
Enlarge this image.Figure 3. Analysis of proteins’ conserved motifs and gene structure of DXS gene family in tomatoes. (A) Gene structure. (B) Protein conserved motifs. (C) Protein conservative domain.
Enlarge this image.Figure 4. Distribution of cis-acting elements in the tomato DXS gene family promoter sequence.
Enlarge this image.Figure 5. Expression patterns of tomato DXS gene under abiotic stress. Treatments with (A) 100 μΜ of MeJA; (B) 100 μΜ of GA; (C) 100 μΜ of ABA; and (D) 100 mM of NaCl. The Actin gene was used as the reference gene, and the expression data were used for the RT-qPCR. The data represent the mean of triplicates with three biological replicates (Supplementary Materials, Figure S1).
Enlarge this image.Figure 6. Analysis of silenced tomato plants. (a) Phenotype of plants silenced by tomato SlDXS genes. (A–D) are phenotypes from 7 d after infestation and (E–H) are phenotypes from one month after infestation. (b) Analysis of tomato TRV2-SlDXS gene expression. (c) Content of chlorophyll a. (d) Content of chlorophyll b. (e) Carotenoid content. The Actin gene was used as the reference gene for (b), and the expression data for the RT-qPCR. The data represent the mean of triplicates with three biological replicates. Error bars represent the standard errors (SEs). Different lowercase letters represent significant differences (p < 0.05).
Enlarge this image.Figure 7. Analysis of TRV2-SlDXS2 gene response to MeJA. (A) Analysis of gene expression in silenced plants in response to MeJA. (B) Flavonoid content of silenced plants. The data represent the mean of triplicates with three biological replicates. Error bars represent the standard errors (SEs). Different lowercase letters represent significant differences (p < 0.05).
Sequence characteristics of the SlDXS proteins.
| Gene Name | Gene ID | AA 1 | Mw 2 | pI 3 | II 4 | Gravy 5 | Subcellular |
|---|---|---|---|---|---|---|---|
| SlDXS1 | 01g067890 | 719 | 77.60 | 6.32 | 40.36 | −0.063 | chloroplast |
| SlDXS2 | 11g010850 | 714 | 77.08 | 6.61 | 40.85 | −0.094 | chloroplast |
| SlDXS3 | 08g066950 | 709 | 77.17 | 5.85 | 35.56 | 0.041 | chloroplast |
1 AA, amino acid; 2 Mw, molecular weight; 3 pI, theoretical isoelectric point; 4 II, instability index; 5 GRAVY, grand average of hydrophobicity.
Secondary structures of proteins in the tomato DXS gene family.
| Gene Name | α-Helix/% | Extended Strand/% | β-Turn/% | Random Coil/% |
|---|---|---|---|---|
| SlDXS1 | 38.94 | 15.30 | 7.51 | 38.25 |
| SlDXS2 | 38.10 | 16.25 | 7.70 | 37.96 |
| SlDXS3 | 41.47 | 14.67 | 7.19 | 36.67 |
Supplementary Materials
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References
1. Dudareva, N.; Klempien, A.; Muhlemann, J.K.; Kaplan, I. Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol.; 2013; 198, pp. 16-32. [DOI: https://dx.doi.org/10.1111/nph.12145]
2. Mahmoud, S.S.; Croteau, R.B. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. Proc. Natl. Acad. Sci. USA; 2001; 98, pp. 8915-8920. [DOI: https://dx.doi.org/10.1073/pnas.141237298]
3. Moses, T.; Pollier, J.; Thevelein, J.M.; Goossens, A. Bioengineering of plant (tri)terpenoids: From metabolic engineering of plants to synthetic biology in vivo and in vitro. New Phytol.; 2013; 200, pp. 27-43. [DOI: https://dx.doi.org/10.1111/nph.12325]
4. Dicke, M.; Sabelis, M.W. How Plants Obtain Predatory Mites as Bodyguards. Neth. J. Zool.; 1987; 38, pp. 148-165. [DOI: https://dx.doi.org/10.1163/156854288X00111]
5. Dicke, M.; Sabelis, M.W.; Takabayashi, J.; Bruin, J.; Posthumus, M.A. Plant strategies of manipulating predatorprey interactions through allelochemicals: Prospects for application in pest control. J. Chem. Ecol.; 1990; 16, pp. 3091-3118. [DOI: https://dx.doi.org/10.1007/BF00979614]
6. Newman, J.D.; Chappell, J. Isoprenoid biosynthesis in plants: Carbon partitioning within the cytoplasmic pathway. Crit. Rev. Biochem. Mol. Biol.; 1999; 34, pp. 95-106. [DOI: https://dx.doi.org/10.1080/10409239991209228]
7. Cordoba, E.; Salmi, M.; León, P. Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. J. Exp. Bot.; 2009; 60, pp. 2933-2943. [DOI: https://dx.doi.org/10.1093/jxb/erp190]
8. Hunter, W.N. The Non-mevalonate Pathway of Isoprenoid Precursor Biosynthesis. J. Biol. Chem.; 2007; 282, pp. 21573-21577. [DOI: https://dx.doi.org/10.1074/jbc.R700005200]
9. Enfissi, E.M.; Fraser, P.D.; Lois, L.M.; Boronat, A.; Schuch, W.; Bramley, P.M. Metabolic engineering of the mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato. Plant Biotechnol. J.; 2005; 3, pp. 17-27. [DOI: https://dx.doi.org/10.1111/j.1467-7652.2004.00091.x]
10. Muñoz-Bertomeu, J.; Arrillaga, I.; Ros, R.; Segura, J. Up-regulation of 1-deoxy-D-xylulose-5-phosphate synthase enhances production of essential oils in transgenic spike lavender. Plant Physiol.; 2006; 142, pp. 890-900. [DOI: https://dx.doi.org/10.1104/pp.106.086355]
11. Gong, Y.F.; Liao, Z.H.; Guo, B.H.; Sun, X.F.; Tang, K.X. Molecular cloning and expression profile analysis of Ginkgo biloba DXS gene encoding 1-deoxy-D-xylulose 5-phosphate synthase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway. Planta Medica; 2006; 72, pp. 329-335. [DOI: https://dx.doi.org/10.1055/s-2005-916234]
12. Zhong, X.; Hou, H.; Qiu, W. Integrity of nonviral fragments in recombinant Tomato bushy stunt virus and defective interfering RNA is influenced by silencing and the type of inserts. Mol. Plant Microbe Interact.; 2005; 18, pp. 800-807. [DOI: https://dx.doi.org/10.1094/MPMI-18-0800]
13. Irmisch, S.; Clavijo McCormick, A.; Günther, J.; Schmidt, A.; Boeckler, G.A.; Gershenzon, J.; Unsicker, S.B.; Köllner, T.G. Herbivore-induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalist caterpillar. Plant J. Cell Mol. Biol.; 2014; 80, pp. 1095-1107. [DOI: https://dx.doi.org/10.1111/tpj.12711]
14. von Mérey, G.E.; Veyrat, N.; D’Alessandro, M.; Turlings, T.C. Herbivore-induced maize leaf volatiles affect attraction and feeding behavior of Spodoptera littoralis caterpillars. Front. Plant Sci.; 2013; 4, 209. [DOI: https://dx.doi.org/10.3389/fpls.2013.00209]
15. Hussain, M.; Debnath, B.; Qasim, M.; Bamisile, B.S.; Islam, W.; Hameed, M.S.; Wang, L.; Qiu, D. Role of Saponins in Plant Defense against Specialist Herbivores. Molecules; 2019; 24, 2067. [DOI: https://dx.doi.org/10.3390/molecules24112067]
16. War, A.R.; Paulraj, M.G.; Ignacimuthu, S.; Sharma, H.C. Induced resistance to Helicoverpa armigera through exogenous application of jasmonic acid and salicylic acid in groundnut, Arachis hypogaea. Pest Manag. Sci.; 2015; 71, pp. 72-82. [DOI: https://dx.doi.org/10.1002/ps.3764]
17. Naranjo-Guevara, N.; Peñaflor, M.; Cabezas-Guerrero, M.F.; Bento, J.M.S. Nocturnal herbivore-induced plant volatiles attract the generalist predatory earwig Doru luteipes Scudder. Sci. Nat.; 2017; 104, 77. [DOI: https://dx.doi.org/10.1007/s00114-017-1498-9]
18. Fürstenberg-Hägg, J.; Zagrobelny, M.; Bak, S. Plant defense against insect herbivores. Int. J. Mol. Sci.; 2013; 14, pp. 10242-10297. [DOI: https://dx.doi.org/10.3390/ijms140510242]
19. Tholl, D. Biosynthesis and biological functions of terpenoids in plants. Adv. Biochem. Eng. Biotechnol.; 2015; 148, pp. 63-106. [DOI: https://dx.doi.org/10.1007/10_2014_295]
20. Finn, R.D.; Coggill, P.; Eberhardt, R.Y.; Eddy, S.R.; Mistry, J.; Mitchell, A.L.; Potter, S.C.; Punta, M.; Qureshi, M.; Sangrador-Vegas, A. et al. The Pfam protein families database: Towards a more sustainable future. Nucleic Acids Res.; 2016; 44, pp. D279-D285. [DOI: https://dx.doi.org/10.1093/nar/gkv1344]
21. Finn, R.D.; Clements, J.; Eddy, S.R. HMMER web server: Interactive sequence similarity searching. Nucleic Acids Res.; 2011; 39, pp. W29-W37. [DOI: https://dx.doi.org/10.1093/nar/gkr367]
22. Marchler-Bauer, A.; Zheng, C.; Chitsaz, F.; Derbyshire, M.K.; Geer, L.Y.; Geer, R.C.; Gonzales, N.R.; Gwadz, M.; Hurwitz, D.I.; Lanczycki, C.J. et al. CDD: Conserved domains and protein three-dimensional structure. Nucleic Acids Res.; 2013; 41, pp. D348-D352. [DOI: https://dx.doi.org/10.1093/nar/gks1243]
23. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant; 2020; 13, pp. 1194-1202. [DOI: https://dx.doi.org/10.1016/j.molp.2020.06.009]
24. Arocho, A.; Chen, B.; Ladanyi, M.; Pan, Q. Validation of the 2−ΔΔCt Calculation as an Alternate Method of Data Analysis for Quantitative PCR of BCR-ABL P210 Transcripts. Diagn. Mol. Pathol.; 2006; 15, pp. 56-61. [DOI: https://dx.doi.org/10.1097/00019606-200603000-00009]
25. Han, Y.; Chen, Z.; Lv, S.; Ning, K.; Ji, X.; Liu, X.; Wang, Q.; Liu, R.; Fan, S.; Zhang, X. MADS-Box Genes and Gibberellins Regulate Bolting in Lettuce (Lactuca sativa L.). Front. Plant Sci.; 2016; 7, 1889. [DOI: https://dx.doi.org/10.3389/fpls.2016.01889] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28018414]
26. Di, X.; Rodriguez-Concepcion, M. Exploring the Deoxy-D-xylulose-5-phosphate Synthase Gene Family in Tomato (Solanum lycopersicum). Plants; 2023; 12, 3886. [DOI: https://dx.doi.org/10.3390/plants12223886]
27. Dudareva, N.; Pichersky, E.; Gershenzon, J. Biochemistry of plant volatiles. Plant Physiol.; 2004; 135, pp. 1893-1902. [DOI: https://dx.doi.org/10.1104/pp.104.049981]
28. Holopainen, J.K.; Blande, J.D. Where do herbivore-induced plant volatiles go?. Front. Plant Sci.; 2013; 4, 185. [DOI: https://dx.doi.org/10.3389/fpls.2013.00185]
29. Tong, Y.; Su, P.; Zhao, Y.; Zhang, M.; Wang, X.; Liu, Y.; Zhang, X.; Gao, W.; Huang, L. Molecular Cloning and Characterization of DXS and DXR Genes in the Terpenoid Biosynthetic Pathway of Tripterygium wilfordii. Int. J. Mol. Sci.; 2015; 16, pp. 25516-25535. [DOI: https://dx.doi.org/10.3390/ijms161025516]
30. Sun, R.; Liu, S.; Gao, J.L.; Tang, Z.Z.; Wu, Q. Cloning and expression analysis of 1-deoxy-D-xylulose-5-phosphate synthase gene from the medicinal plant Conyza blinii H. Lév. Turk. J. Biol.; 2015; 38, pp. 664-670. [DOI: https://dx.doi.org/10.3906/biy-1401-91]
31. Zhou, W.; Huang, F.; Li, S.; Wang, Y.; Zhou, C.; Shi, M.; Wang, J.; Chen, Y.; Wang, Y.; Wang, H. et al. Molecular cloning and characterization of two 1-deoxy-d-xylulose-5-phosphate synthase genes involved in tanshinone biosynthesis in Salvia miltiorrhiza. Mol. Breed.; 2016; 36, 124. [DOI: https://dx.doi.org/10.1007/s11032-016-0550-3]
32. Carretero-Paulet, L.; Cairó, A.; Talavera, D.; Saura, A.; Imperial, S.; Rodríguez-Concepción, M.; Campos, N.; Boronat, A. Functional and evolutionary analysis of DXL1, a non-essential gene encoding a 1-deoxy-D-xylulose 5-phosphate synthase like protein in Arabidopsis thaliana. Gene; 2013; 524, pp. 40-53. [DOI: https://dx.doi.org/10.1016/j.gene.2012.10.071]
33. Cordoba, E.; Porta, H.; Arroyo, A.; San Román, C.; Medina, L.; Rodríguez-Concepción, M.; León, P. Functional characterization of the three genes encoding 1-deoxy-D-xylulose 5-phosphate synthase in maize. J. Exp. Bot.; 2011; 62, pp. 2023-2038. [DOI: https://dx.doi.org/10.1093/jxb/erq393]
34. Liu, W.; Wang, H.; Chen, Y.; Zhu, S.; Chen, M.; Lan, X.; Chen, G.; Liao, Z. Cold stress improves the production of artemisinin depending on the increase in endogenous jasmonate. Biotechnol. Appl. Biochem.; 2017; 64, pp. 305-314. [DOI: https://dx.doi.org/10.1002/bab.1493]
35. de Luna-Valdez, L.; Chenge-Espinosa, M.; Hernández-Muñoz, A.; Cordoba, E.; López-Leal, G.; Castillo-Ramírez, S.; León, P. Reassessing the evolution of the 1-deoxy-D-xylulose 5-phosphate synthase family suggests a possible novel function for the DXS class 3 proteins. Plant Sci.; 2021; 310, 110960. [DOI: https://dx.doi.org/10.1016/j.plantsci.2021.110960] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/34315585]
36. Zhang, S.; Ding, G.; He, W.; Liu, K.; Luo, Y.; Tang, J.; He, N. Functional Characterization of the 1-Deoxy-D-Xylulose 5-Phosphate Synthase Genes in Morus notabilis. Front. Plant Sci.; 2020; 11, 1142. [DOI: https://dx.doi.org/10.3389/fpls.2020.01142] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32849701]
37. Hao-Yu, Z.; Jun-Miao, F.; Ting, W.; Yuan-Huai, H.; Fang, D. Advances on Key Gene DXS Involved in the Terpenoid Biosynthesis in Plants. Biotechnol. Bull.; 2018; 34, pp. 1-8. [DOI: https://dx.doi.org/10.13560/j.cnki.biotech.bull.1985.2017-0779]
38. Walter, M.H.; Hans, J.; Strack, D. Two distantly related genes encoding 1-deoxy-d-xylulose 5-phosphate synthases: Differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots. Plant J. Cell Mol. Biol.; 2002; 31, pp. 243-254. [DOI: https://dx.doi.org/10.1046/j.1365-313X.2002.01352.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/12164805]
39. Floss, D.S.; Hause, B.; Lange, P.R.; Küster, H.; Strack, D.; Walter, M.H. Knock-down of the MEP pathway isogene 1-deoxy-D-xylulose 5-phosphate synthase 2 inhibits formation of arbuscular mycorrhiza-induced apocarotenoids, and abolishes normal expression of mycorrhiza-specific plant marker genes. Plant J. Cell Mol. Biol.; 2008; 56, pp. 86-100. [DOI: https://dx.doi.org/10.1111/j.1365-313X.2008.03575.x] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18557838]
40. Esteban, R.; Barrutia, O.; Artetxe, U.; Fernández-Marín, B.; Hernández, A.; García-Plazaola, J.I. Internal and external factors affecting photosynthetic pigment composition in plants: A meta-analytical approach. New Phytol.; 2015; 206, pp. 268-280. [DOI: https://dx.doi.org/10.1111/nph.13186] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25414007]
41. Zhang, B.; Bai, Y.; Chen, J. Effects of Photosynthesis by Pests and Diseases: A Review. J. Henan Norm. Univ. Nat. Sci. Ed.; 2015; 43, pp. 119-125.
42. Peng, G.; Wang, C.; Song, S.; Fu, X.; Azam, M.; Grierson, D.; Xu, C. The role of 1-deoxy-d-xylulose-5-phosphate synthase and phytoene synthase gene family in citrus carotenoid accumulation. Plant Physiol. Biochem. PPB; 2013; 71, pp. 67-76. [DOI: https://dx.doi.org/10.1016/j.plaphy.2013.06.031]
43. Estévez, J.M.; Cantero, A.; Reindl, A.; Reichler, S.; León, P. 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J. Biol. Chem.; 2001; 276, pp. 22901-22909. [DOI: https://dx.doi.org/10.1074/jbc.M100854200] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11264287]
44. Zhang, M.; Li, K.; Zhang, C.; Gai, J.; Yu, D. Identification and characterization of class 1 DXS gene encoding 1-deoxy-D-xylulose-5-phosphate synthase, the first committed enzyme of the MEP pathway from soybean. Mol. Biol. Rep.; 2009; 36, pp. 879-887. [DOI: https://dx.doi.org/10.1007/s11033-008-9258-8] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18437529]
45. Wei, H.; Movahedi, A.; Xu, C.; Sun, W.; Almasi Zadeh Yaghuti, A.; Wang, P.; Li, D.; Zhuge, Q. Overexpression of PtDXS Enhances Stress Resistance in Poplars. Int. J. Mol. Sci.; 2019; 20, 1669. [DOI: https://dx.doi.org/10.3390/ijms20071669] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30987184]
46. Li, R.; Chen, P.; Zhu, L.; Wu, F.; Chen, Y.; Zhu, P.; Ji, K. Characterization and Function of the 1-Deoxy-D-xylose-5-Phosphate Synthase (DXS) Gene Related to Terpenoid Synthesis in Pinus massoniana. Int. J. Mol. Sci.; 2021; 22, 848. [DOI: https://dx.doi.org/10.3390/ijms22020848] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33467778]
47. Ya-Fei, G.; Jun-Ya, W.; Fei, G.; De-Jiang, N. Cloning and Expression Analysis of CsDXS1 Gene Encoding 1-Deoxy-D-Xylulose-5-Phosphate Synthase in Camellia sinensis. Biotechnol. Bull.; 2018; 34, pp. 144-152. [DOI: https://dx.doi.org/10.13560/j.cnki.biotech.bull.1985.2017-0652]
48. Li, W.; Li, W.; Yang, S.; Ma, Z.; Zhou, Q.; Mao, J.; Han, S.; Chen, B. Transcriptome and Metabolite Conjoint Analysis Reveals that Exogenous Methyl Jasmonate Regulates Monoterpene Synthesis in Grape Berry Skin. J. Agric. Food Chem.; 2020; 68, pp. 5270-5281. [DOI: https://dx.doi.org/10.1021/acs.jafc.0c00476]
49. Li, J.; Yu, H.; Liu, M.; Chen, B.; Dong, N.; Chang, X.; Wang, J.; Xing, S.; Peng, H.; Zha, L. et al. Transcriptome-wide identification of WRKY transcription factors and their expression profiles in response to methyl jasmonate in Platycodon grandiflorus. Plant Signal. Behav.; 2022; 17, 2089473. [DOI: https://dx.doi.org/10.1080/15592324.2022.2089473]
50. Dong, Y.; Li, J.; Zhang, W.; Bai, H.; Li, H.; Shi, L. Exogenous application of methyl jasmonate affects the emissions of volatile compounds in lavender (Lavandula angustifolia). Plant Physiol. Biochem.; 2022; 185, pp. 25-34. [DOI: https://dx.doi.org/10.1016/j.plaphy.2022.05.022]
51. Yao, S.; Cao, S.; Hou, S.; Huang, F.; Mo, G.; Han, L. Effects of Methyl Jasmonate on the Accumulation of Triterpenoid Saponin Anhuienoside E in Anemone flaccida. J. Hubei Univ. Chin. Med.; 2019; 21, pp. 38-41.
52. Hoffmann, J.A.; Kafatos, F.C.; Janeway, C.A.; Ezekowitz, R.A. Phylogenetic perspectives in innate immunity. Science; 1999; 284, pp. 1313-1318. [DOI: https://dx.doi.org/10.1126/science.284.5418.1313] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10334979]
53. Paetzold, H.; Garms, S.; Bartram, S.; Wieczorek, J.; Urós-Gracia, E.M.; Rodríguez-Concepción, M.; Boland, W.; Strack, D.; Hause, B.; Walter, M.H. The isogene 1-deoxy-D-xylulose 5-phosphate synthase 2 controls isoprenoid profiles, precursor pathway allocation, and density of tomato trichomes. Mol. Plant; 2010; 3, pp. 904-916. [DOI: https://dx.doi.org/10.1093/mp/ssq032] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20591838]
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