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Promoter characterization of a citrus linalool synthase gene mediating interspecific variation in resistance to a bacterial pathogen



Terpenoids play essential roles in plant defense against biotic stresses. In Citrus species, the monoterpene linalool mediates resistance against citrus canker disease caused by the gram-negative bacteria Xanthomonas citri subsp. citri (Xcc). Previous work had associated linalool contents with resistance; here we characterize transcriptional responses of linalool synthase genes.


Leaf linalool contents are highly variable among different Citrus species. “Dongfang” tangerine (Citrus reticulata), a species with high linalool levels was more resistant to Xcc than “Shatian” pummelo (C. grandis) which accumulates only small amounts of linalool. The coding sequences of the major leaf-expressed linalool synthase gene (STS4) are highly conserved, while transcript levels differ between the two Citrus species. To understand this apparent differential transcription, we isolated the promoters of STS4 from the two species, fused them to a GUS reporter and expressed them in Arabidopsis. This reporter system revealed that the two promoters have different constitutive activities, mainly in trichomes. Interestingly, both linalool contents and STS4 transcript levels are insensitive to Xcc infestation in citrus plants, but in these transgenic Arabidopsis plants, the promoters are activated by challenge of a bacterial pathogen Pseudomonas syringae, as well as wounding and external jasmonic acid treatment.


Our study reveals variation in linalool and resistance to Xcc in citrus plants, which may be mediated by different promoter activities of a terpene synthase gene in different Citrus species.

Peer Review reports


Citrus is the largest fruit category in the world. Citrus canker is a devastating disease threatening the citrus industry worldwide. This disease is caused by a Gram-negative bacterium Xanthomonas citri subsp.citri (Xcc) [1, 2]. Research into citrus canker has been focused on the pathogen’s genome, host-pathogen interactions, resistant or susceptible genes of host. For example, Da Silva et al. [3] sequenced the genome of Xcc-A306 strain, and found a large number of genes coding for cell wall degrading enzymes (CWDEs), proteases, type 2 secretion system (T2SS) and type 3 secretion system (T3SS). Whereas Zou et al. [4] identified the susceptibility gene CsLOB1 in Citrus plants.

Plants produce a large number of specialized metabolites to defend themselves from environmental stresses such as attack from herbivores and pathogens [5,6,7]. The largest sector of these metabolites are the terpenoids, which are composed of isoprenoid units with highly diverse structures [8]. Thousands of terpenoid structures have been described. Based on the number of isoprenoid units in the molecules, terpenoids are classified as hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), tetraterpenes (C40), and their derivatives [9,10,11]. Hemi-, mono-, sesqui- and a few di-terpenes can be emitted to headspace as volatiles from particular organs, such as flowers, or after environmental stimulation such as oviposition, herbivory or wounding [12,13,14]. Di-, tri-, and tetraterpenes are mostly non-volatile compounds which increase in organs in response to (a)biotic stresses [15,16,17].

Volatile terpenoids are usually synthesized in plants through two pathways: the 2-C-methyl-D-erythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway in plastids and the mevalonate pathway in the cytosol and peroxisomes [18, 19]. Terpene synthases (TPS) play important roles in these pathways by converting the precursor, geranyl diphosphate, into monoterpenes, farnesyl diphosphate into sesquiterpenes and geranylgeranyl diphosphate into diterpenes. TPS genes are found throughout the plant kingdom, from mosses to flower plants, and usually share structures and comprise medium-sized gene family, commonly divided into seven major branches from TPS-a to TPS-g by phylogenetic analysis[20]. For example, 40 TPSs were identified in the Arabidopsis thaliana genome [20], 27 in Cucumis sativa [21], and 152 in Vitis vinifera [22]. In Citrus, Dornelas et al. [23] found 49 TPSs by searching CitEST database. Whereas from genome sequences, 95 TPSs were identified in Citrus sinensis [24] and 58 in Finger citron (C. medica var. sarcodactylis) [25]. Like other terpenoids that respond to biotic stress, many TPSs are upregulated by herbivory [26,27,28,29,30,31].

Many terpenoids and TPSs are involved in the interactions between plants and their environment. For example, oviposition and injury by herbivores frequently elicits the release of terpenoid volatiles [13, 14, 16]. Light is also known to regulate terpenoids releases in several species [32]. Promoter regions of TPS genes have been analyzed and many stress-related cis-acting regulatory elements such as G-box elements, W-boxes elements, ABRE motifs and MYB binding sites are commonly found. The G-box elements are required for JA-mediated transcription regulation, which may be involved in herbivory responses, as well as being essential for light regulation [33]. W-boxes are associated with SA responses and ABRE motifs are associated with ABA responses [32]. W-boxes and MYB binding sites can specifically bind WRKY and MYB transcription factors that play important roles in disease resistance [34, 35].

Linalool is a monoterpene alcohol found in many plant species and has many different functions. It is a common component of floral scents [36], known to attract pollinators. In some cases, linalool and its derivatives can also act as an insect repellent [37, 38]. Linalool is also a volatile emitted from leaves. For example, in tomato, it mainly accumulates in trichomes [39]. Linalool is also found in insects where it may function as a pheromone [40] and a pathogen defense compound [41]. It is also in many natural essential oils, valued for its antibacterial activity against many gram-negative bacteria, with promise as a medicinal therapeutic [42]. In summary, linalool is widely found in the biological world with different context-dependent functions in interacting systems [43].

Citrus plants are often richly endowed in volatiles, among which linalool, limonene, caryophyllene and other terpenes are dominant components [44]. Shimada et al. [45] isolated and identified cDNAs of several monoterpene synthase genes including three linalool synthases from C. unshiu. Over-expression of citrus linalool synthase gene in sweet orange, which is susceptible to canker disease, enhanced its resistance to the pathogen [46]. Furthermore, linalool treatment inhibits the growth of Xcc in vitro [45], suggesting that linalool may function in citrus defense against Xcc. Given the context-dependence of linalool function, it is surprising that little is known about the transcriptional regulation of linalool synthase genes.

Here, we found that leaf linalool content varies greatly amongst 6 Citrus species and analyzed the promotor sequences both in silico and in an Arabidopsis reporter system, of two linalool synthase genes (STS4) from two Citrus species that divergently accumulate linalool and differ in Xcc resistance: C. reticulata and C. grandis.


C. reticulata has higher leaf linalool contents than C. grandis andC. medica, and stronger Xcc resistance

To investigate the variation of foliar linalool contents, six Citrus accessions including 2 C. grandis, 2 C. medica and 2 C. reticulata were selected to measure the internal linalool content in their leaves. We found that linalool contents in C. reticulata leaves were much higher than that in C. grandis and C. medica leaves, about 8–42 fold higher (Fig. 1a).

Fig. 1
figure 1

Variation of linalool in Citrus species and resistance against canker disease. (a) Relative linalool abundance in leaves of six Citrus cultivars including two C. reticulata, two C. grandis and two C. medica. The linalool content is much higher in two C. reticulata cultivars than in cultivars of the other two specie (n = 3, p < 0.05, ANOVA) s. (b) Leaves of a C. reticulata (“Dongfang” tangerine) and a C. grandis (“Shatian” pummelo) infected by Xanthomonas citri subsp.citri. Shown are representative leaves from experiments repeated three times, with at least three replicates in each treatment group. (c) Colony forming units (cfu) were counted at seven sites to assess the accumulation of bacterial populations (n = 3, * * p < 0.01, t-test)

Previous studies showed that linalool in citrus plants may mediate resistance against Xanthomonas citri subsp.citri (Xcc). We infested “Dongfang” tangerine (high linalool) and “Shatian” pummelo (low linalool) with Xcc. Ten days after infestation of the pathogen, “Dongfang” tangerine showed smaller sponge lesions and fewer colonies (Fig. 1b 1c) than did “Shatian” pummelo, indicating stronger resistance to Xcc.

A linalool synthase gene is only highly transcribed in C. reticulata

Shimada et al. [45] identified three linalool synthase genes STS4, STS3-1 and STS3-2 in citrus. In order to study which genes may control linalool levels in leaves, we performed qPCR analysis of these three genes in six varieties (Fig. 2a, Fig. S1), finding that STS4 show the highest expression level in leaves. Combined with linalool measurement results shown previously, we selected the representative genotypes “Dongfang” tangerine and “Shatian” pummelo for subsequent research into the role of STS4 in resistance to Xcc.

Fig. 2
figure 2

Variation in transcript levels of linalool synthase genes and in coding sequences of the major leaf-expressed STS4in citrus species. (a) Relative transcript abundances of three linalool synthase genes in leaves of different citrus cultivars (n = 3, p < 0.05, ANOVA). (b) Alignment of amino acid sequences encoded by the orthologs of the major leaf-expressed STS4 between C. reticulata (CrSTS4) and C. grandis (CgSTS4). Typical terpene synthase motifs are indicated in red. (c) Phylogenetic relationship between linalool synthase gene and plant terpene synthases (TPSs) in CrSTS4 and CgSTS4. The tree was constructed using the neighbor-joining (NJ) method

We isolated the cDNA of this gene from leaves of “Dongfang” tangerine and “Shatian” pummelo (CrSTS4 and CgSTS4, respectively). The amino acid sequence alignment of CrSTS4 and CgSTS4 showed that they were highly similar (98.8%). The typical motifs for active mono-TPS genes such as RR(x)8 W and DDxxD are present in both genes (Fig. 2b). A phylogenic analysis of the 2 STS4 genes among previously characterized TPSs revealed that both genes were in the TPS-a subfamily, and the closest gene was the linalool synthase gene CuSTS4 identified in C. unshiu (Fig. 2c).

STS4 promoter regions differ between C. reticulata and C. grandis and harbor multiple stress-related cis-acting elements

To reveal the mechanism responsible for the different accumulations of CrSTS4 and CgSTS4 transcripts, we sequenced 1999 and 2152 bp upstream of the start codons of CrSTS4 and CgSTS4, respectively. Alignment of these promoter sequences revealed an identity of 93.89% (Fig. 3a), and a number of SNPs and insertion/deletion variations between them. Remarkably, a 137 bp sequence was only present in the promoter region of C. grandis.

Fig. 3
figure 3

Analysis of STS4 promoter sequences in C. reticulata and C. grandis. (a) Nucleic acid sequence alignment of pCrSTS4 (promoter of STS4 isolated from C. reticulata) and pCgSTS4 (promoter of STS4 isolated from C. grandis). The different areas are in red. (b) Analysis of putative cis-acting elements in CrSTS4 and CgSTS4. Putative functions of the motifs are indicated by different colors

We analyzed the potential cis-regulatory elements of the two promoters using PlantCARE (Fig. 3b, Table S1). The CrSTS4 and CgSTS4 promoters shared most of the elements which might be regulated by environmental factors such as light, hormones and mechanical damage. The CgSTS4 promoter has an AAGAA-motif element and a GATA-motif element lacking in the CrSTS4 promoter, in addition to MYB and MYC elements, implying that the CgSTS4 gene may be more strongly regulated by environmental stresses.

In transgenic Arabidopsis, the CrSTS4 promoter showed stronger constitutive activity in trichomes

In order to reveal the activity of the promoters of CrSTS4 (pCrSTS4) and CgSTS4 (pCgSTS4), we fused them with a GUS reporter gene and transformed them into Arabidopsis. GUS-staining of the rosette leaves revealed that the GUS reporter driven by pCrSTS4 was much more strongly expressed than that driven by pCgSTS4 (Fig. 4a 4b).

Fig. 4
figure 4

pCrSTS4 exhibits stronger constitutive activity than pCgSTS4 after transformation into Arabidopsis, specifically in veins and trichomes. (a)GUS staining analysis of the third generation homozygous transgenic Arabidopsis thaliana harboring pCrSTS4::GUS or pCgSTS4::GUS (n = 3, * * *p < 0.001, t-test). (b) The transcript abundance of the GUS gene in transgenic Arabidopsis plants. (c) Both pCrSTS4 and pCgSTS4 were active in the trichomes in the transgenic Arabidopsis, pCrSTS4 was also active in the veins. (d) GUS-stained tobacco leaves transiently expressing pCrSTS4 and pCgSTS4. Similar expression patterns were present for both promoters. c) and d) were observed with an optical microscope

In order to reveal the spatial expression of CrSTS4 and CgSTS4, the GUS-stained Arabidopsis leaves were observed under an optical microscope. In both pCrSTS4::GUS and pCgSTS4::GUS transgenic plants, the trichomes were strongly stained. Whereas veins and mesophyll cells were also stained only in pCrSTS4::GUS plants (Fig. 4c).

We further assessed spatial expression by transiently expression of pCrSTS4::GUS and pCgSTS4::GUS constructs in cultivated tobacco leaves using Agrobacterium infiltration. Staining of the infiltrated leaves revealed a similar spatial expression as in Arabidopsis, being strong in the trichomes (Fig. 4d).

Pst DC3000 and phytohormone elicitation of CrSTS4 and CgSTS4 expression in transgenic Arabidopsis

Many terpenoids are known to be induced by biological stresses such as herbivory or pathogen attack. We tested if linalool in “Dongfang” tangerine and “Shatian” pummelo was inducible upon infestation of Xcc. Three days after Xcc inoculation, both tangerine and pummelo showed no difference in linalool contents compared to the controls (Fig. 5a). Similar results were found with Mangshanyeju (C. reticulata) and kumquat (Poncirus) (Fig. S2a). Consistently, the transcript abundance of the STS4 gene in these varieties was also not influenced by the elicitations (Fig. 5b, Fig. S2b). However, since we found a number of stress related cis-acting elements present in their promoter regions, we tested the transgenic Arabidopsis plants for their responses to the bacterial pathogen Pst DC3000. Interestingly, GUS-staining revealed that both pCrSTS4 and pCgSTS4 were strongly activated (Fig. 5c). We further found that the activities of the two promoters were also induced by mechanic wounding and exogenous treatment with JA. However, only pCrSTS4 responded to gibberellin (GA3) and abscisic acid (ABA) treatments (Fig. 5c).

Fig. 5
figure 5

Linalool and transcription of STS4 in citrus do not response to Xcc infection but pCrSTS4 and pCgSTS4 are activated by infection with bacterial pathogen, wounding and phytohormones in transgenic Arabidopsis. (a) Relative abundance of linalool in leaves of C. reticulata and C. grandis plants with or without Xcc infection (n = 3, t-test). (b) Transcript levels of CrSTS4 and CgSTS4 in leaves of infected or noninfected citrus plants, measured with qRT-PCR (n = 3, t-test). (c) GUS-stained leaves of transgenic Arabidopsis plants harboring pCrSTS4 or pCgSTS4 challenged by Pst DC3000, mechanical damage, and treatment of external GA, ABA and JA


Linalool is present in more than 200 monocotyledonous and dicotyledonous plants. In particular, many plants in Labiatae, Lauraceae and Rutaceae contain large amounts of linalool. We found considerable variation in foliar linalool contents in different Citrus species. Citrus reticulata, such as “Shatang” and “Dongfang” tangerine contains more linalool than C. grandis accessions such as “Fenghuang” and “Shatian” pummelo and C. medica accessions such “Nanchuan” and “Danna” citron. Linalool is an important defensive metabolite. As a volatile cue, it can directly repel some insects [37], and mediate tri-trophic interactions between plants, herbivores and natural enemies [38, 43]. Moreover, linalool has broad-spectrum resistance to a variety of pathogenic microorganisms [47], including a variety of human oral bacterial pathogens [48] and the pathogenic fungus Alternaria alternata [49]. Droby et al. [50] reported that linalool inhibits the germination of Penicillium italicum and P. digitatum spores. And linalool was found to have the antibacterial activity against Acinetobacter baumanni [51]. In citrus, linalool inhibits the growth of Xcc and P. italicum, the pathogen of postharvest rot disease of citrus [45]. Furthermore, in different citrus varieties the content of linalool appears to be associated with resistance to Xcc [52]. Overexpression of a linalool synthase gene in the citrus variety “Hamlin” sweet orange increased resistance to canker disease [46]. In our study, “Dongfang” tangerine, a variety with high linalool abundance, showed stronger resistance to Xcc than “Shatian” pummelo, both in lesion size and colony statistics. A previous study which compared long-term canker-resistance among 186 citrus genotypes in the field found that many tangerine (C. reticulata) genotypes were among the most resistant ones [53]. On the other hand, even though the C. grandis genotypes were not included in this study, its close relative, C. paradise are among the most susceptible genotypes of this study. This difference in resistance may be due to many factors including cuticle thickness of the leaf, wax content, stomata density and linalool content, as revealed in this study.

Terpene synthase (TPS) is responsible for the synthesis of various terpene molecules from precursors. Intra-specific variation of terpenoids could be caused by the variation in responsible terpene synthase in different varieties of same species or closely related species. For example, we previously found that in different varieties of the wild tobacco, Nicotiana attenuata, a linalool synthase has two alleles. One allele encodes an enzyme with full efficiency to synthesize linalool, while the other allele harbors a deletion in the coding sequence and is not correctly spliced, to encode an enzyme inefficient in synthesizing linalool. This allelic variation accounts the differences in linalool accumulation among geographically interspersed conspecific wild tobacco plants [38]. However, in this study, variation in linalool among Citrus. spp seems to be controlled by the different STS4 transcript abundances levels. This inference is based on the observations that the isolated CDS of CrSTS4 and CgSTS4 harbored only minor variations and both encoded enzymes with fully functional TPS domains. Moreover, CrSTS4 transcript levels in tangerine are much higher than those of CgSTS4 in pummelo, which is consistent with their linalool contents. We found larger differences in the promoter regions than that in coding sequence of STS4 between the two Citrus species. Although we can not exclude the possibility that the minor variation in coding sequences of CrSTS4 and CgSTS4 could account for differences in the rates of linalool biosynthesis, the available data is consistent with transcriptional regulation of linalool. This inference is also consistent with the observation that pCrSTS4 exhibited stronger activity than pCgSTS4 when transformed into Arabidopsis.

Both pCrSTS4 and pCgSTS4 are specifically active in trichomes, both in Arabidopsis and in tobacco. The trichomes are the first line of defense against stress in plants, and trichomes can directly sense external mechanical forces to predict pathogen infection [54]. However, there are no trichome structure on citrus leaves, which instead have thick oil glands that resist stress and pathogen invasion [55]. Some TPS genes have been found to be specifically transcribed in the epithelial cells surrounding the oil glands in rough lemon leaf [56] and it is likely that STS4 is also expressed in a similar tissue. However, additional experiments in citrus are required to verify this hypothesis.

Many terpenoids and their synthase genes are responsive to environmental stresses such as herbivores, pathogens, and mechanical damage or external methyl jasmonate(MeJA. This kind of induction has been reported in a number of plant species including conifers [57, 58], tomato [28, 29], maize [30], leguminous plants [26] and cucumber [32]. In our study the variation of linalool and transcription of STS4 are constitutive present in the Citrus spp. Previous study showed that STS4 in C. unshiu was upregulated by infestation of Xcc [45]. However, later study from the same laboratory reported conflicting results [52]. We found a number of stress-related or hormone-responsive cis-acting regulatory elements present in the promoters of STS4 isolated from two citrus species (pCrSTS4 and pCgSTS4), but did not find significant alternations in linalool abundance in both tangerine and pummelo after infestation of Xcc. Consistently, transcript levels of STS4 in the two species were also not induced by Xcc. Interestingly, pCrSTS4 and pCgSTS4 transferred into Arabidopsis were significantly activated by infection of a bacterial pathogen Pst DC3000. This could be because STS4 is a defense-related gene and is generally responsive to invading pathogens. However, this general ability could be inactivated in a specific susceptible interaction between Citrus plants and Xcc. Furthermore, we found that pCrSTS4 and pCgSTS4 were also activated by mechanical damage and external JA, implying that STS4 might also play a role in defense against other stresses such as herbivory, responses which are commonly regulated by the JA signaling pathway.


In conclusion, this study found that linalool content in tangerine leaves is higher than in other citrus species and has stronger resistance to Xcc. This high level of linalool is associated with higher transcript levels of a linalool synthase gene. The promoter of this gene from tangerine shows stronger activities than that from pummelo, after being transformed into Arabidopsis. Interestingly, although this gene is not induced by Xcc infection, its promoter is activated by Pst DC3000 in transgenic Arabidopsis thaliana. This study provides insights into how constitutive and induced terpene synthase genes combat bacterial pathogens in Citrus, information which could be useful for breeding resistant varieties of Citrus.

Materials and methods

Plant material and pathogen inoculation

“Dongfang” tangerine (C. reticulata), “Shatian” pummelo (C. grandis) and other plant samples used in this study were from the National Citrus Germplasm Repository in Chongqing, China. Xcc was from a culture maintained in our laboratory. The leaves were surface-sterilized with 75% ethanol on a sterile bench before inoculation with Xcc. A wound in the leaf lamina was created using a needle (0.5 mm). The double distilled water re-suspended Xcc suspension (OD600 = 0.6) was injected to about 3/4 of the leaf area; water was injected as a control. After inoculation, leaves were cultured on a sterile petri dish and the petioles were wetted. After 3 days of culture in the incubator (28℃, 60% humidity), the injected area of leaf was taken for subsequent experiments. Each experiment included three biological replicates.

Using the same materials, wounds were created with needles (0.5 mm), and inoculated with 1 µl of each Xcc suspension (OD600 = 0.6). Ulcer symptoms were imaged at 10 days after inoculation. Three lesions were thoroughly ground in double distilled water. After continuous dilution, the liquid was spread on an LB solid plate and cultured at 28 °C for 3 days. The colony forming units of the three lesions were counted.

The Pst DC3000 pathogen challenge was performed with the transgenic Arabidopsis harboring promoter regions of CrSTS4 and CgSTS4 following the method of Fang et al. [59] with minor modifications. Pst DC3000 was cultured in KB medium containing 50 mg/ml rifampicin to OD600 = 0.5. The culture was centrifuged at 5000 g for 5 min, and resuspended in sterile 10 mM MgCl2 buffer to OD600 = 0.0005. Then, the bacteria were inoculated into the adaxial side of 4-week-old homozygous transgenic Arabidopsis thaliana leaves using a needle-free 1 mL syringe, and 10 mM MgCl2 buffer was injected as a control. Each experiment was repeated five times.

GC-MS analysis

One hundred mg of Xcc treated citrus leaves were ground in liquid nitrogen, re-suspended in 5 mL saturated NaCl solution in a glass vial with a stir bar. Cyclohexanone was added to the solution as the internal standard. The vial was sealed with a septum screw-top cap. The samples were equilibrated in a water bath at 40 °C for 20 min, and volatile compounds were collected by solid phase microextraction (SPME) method. A fiber coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, Supelco, Bellefonte, PA) was exposed at the top space of the capped vial for 30 min. The SPME fibers were desorbed for 5 min. The volatiles were determined using an Agilent 7890B gas chromatography and an Agilent 5977 A mass spectrometry. Volatile compounds were identified by comparing its retention time and mass spectrometry matching the mass spectral library (NIST11, W10N14).

Cloning and qRT-PCR analysis of linalool synthase gene

The sequences of linalool synthase genes in tangerine (CrSTS4) and pummelo (CgSTS4) genomes were retrieved using BLAST in Citrus Pan-genome to Breeding Database ( Biospin Plant Total RNA Extraction Kit (BioFlux) was used to extract total RNA from the leaves of “Dongfang” tangerine and “Shatian” pummelo inoculated with Xcc and water controls. cDNA was synthesized and used as template (primers in Table S2) to amplify the linalool synthase genes from the two varieties using the PrimeSTAR Max DNA polymerase (TaKaRa). The obtained PCR product was purified and connected using the 5minTM TA / Blunt-Zero Cloning Kit (Vazyme), and transformed into E.coli Mach1-T1 competent cells for culture and sequencing. The comparison analysis was performed using CLC Sequence Viewer 7.

The cDNA was used as a template, qPCR primers (Table S2) were designed, and CitActin was used as internal reference gene. The cycle procedure was as follows: initial denaturation at 95 °C for 30 s, and then amplification for 40 cycles (95 °C 10 s, 58 °C 30 s, 72 °C 30 s). By associating the Ct value of the expression level with the Ct value of the reference gene CitActin, the cycle threshold (Ct) value of the original data was converted to a standardized expression level by the 2−ΔCt method (27).

Cloning of promoter region and generation of transgenic arabidopsis

In Citrus Pan-genome to Breeding Database (, the 1999 bp (C. reticulata v1.0) and 2152 bp (C. grandis (L.) “ Wanbaiyou” v1.0) fragments upstream of the start codon of CrSTS4 (pCrSTS4) and CgSTS4 (pCgSTS4) were extracted from the genomic sequences for primer design, respectively (Table S2).

By ClonExpress II One Step Cloning Kit (Vazyme) kit, pCrSTS4 and pCgSTS4 were ligated to the pCambia-2016-GUS vector, respectively. A promoter::GUS reporter gene system was constructed and transformed into the E. coli clone strain Mach1-T1 and then into Agrobacterium tumefaciens GV3101 strain. Transformation of Arabidopsis was performed by flower infiltration [60]. The seeds were screened for positive constructs using MS-Kanamycin (50 mg / mL) solid medium.

Promoter cis -acting element analysis

The PlantCARE database ( was used to analyze the cis-acting elements present in pCrSTS4 and pCgSTS4. The alignment motifs of each promoter are listed as their distance from the start codon of the gene.

Activity identification of promoter region and optical microscope observation

Four-week-old T3 homozygous transgenic Arabidopsis thaliana reporter plants were screened. During the active photoperiod, leaves of similar size in the same part of the plants were taken and stained with GUS Stain Kit (Solarbio) at 37 °C for 8 h. The solution discoloration and blue spots on the leaves could be observed by the naked eyes. After 70% alcohol decolorization, leaves were photographed, and placed on a glass slide for examination with an optical microscope.

Elicitation experiments of transgenic arabidopsis

Following the methods of He et al. [32], leaves of the transgenic Arabidopsis plants were challenged by: mechanical damage (using a needle with a diameter of 0.2 mm to puncture the blade); hormone treatments with jasmonic acid(JA), abscisic acid(ABA), and gibberellin (GA3) (all in 5 µl of 1 mM + 0.01% Tween-20), were followed by GUS Stain Kit (Solarbio) staining at 37 °C for 8 h, and 70% alcohol fading for observation and photographing.

Data Availability

The datasets used and/or analyzed during the current study, are available from the corresponding author on reasonable request.


Xcc :

Xanthomonas citri subsp. Citri

Pst DC3000:

Pseudomonas syringae DC3000


Jasmonic acid


Abscisic acid




Methyl jasmonate


Terpene synthase


  1. Omar AA, Murata MM, El-Shamy HA, Graham JH, Grosser JW. Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice. Transgenic Res. 2018;27(2):179–91.

    CAS  PubMed  Google Scholar 

  2. Schaad NW, Postnikova E, Lacy GH, Sechler A, Agarkova I, Stromberg PE, Stromberg VK, Vidaver AK. Reclassification of Xanthomonas campestris pv. citri (ex Hasse 1915) dye 1978 forms a, B/C/D, and E as X. smithii subsp citri (ex Hasse) sp nov nom. rev. comb. nov., X. fuscans subsp aurantifolii (ex Gabriel 1989) sp nov nom. rev. comb. nov., and X. alfalfae subsp citrumelo (ex Riker and Jones) Gabriel et al., 1989 sp nov nom. rev. comb. nov.; X. campestris pv malvacearum (ex Smith 1901) dye 1978 as X. smithii subsp smithii nov comb. nov nom. nov.; X. campestris pv. Alfalfae (ex Riker and Jones, 1935) dye 1978 as X. alfalfae subsp alfalfae (ex Riker et al., 1935) sp nov nom. rev.; and “var. fuscans” of X. campestris pv. Phaseoli (ex Smith, 1987) dye 1978 as X. fuscans subsp fuscans sp nov. Syst Appl Microbiol. 2005;28(6):494–518.

    CAS  PubMed  Google Scholar 

  3. Da Silva AC, Ferro JA, Reinach FC, Farah CS, Furlan LR, Quaggio RB, Monteiro-Vitorello CB, Van Sluys MA, Almeida NF, Alves LM, et al. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature. 2002;417(6887):459–63.

    PubMed  Google Scholar 

  4. Zou XP, Du MX, Liu YN, Wu L, Xu LZ, Long Q, Peng AH, He YR, Andrade M. Chen SC CsLOB1 regulates susceptibility to citrus canker through promoting cell proliferation in citrus. Plant J. 2021;106(4):1039–57.

    CAS  PubMed  Google Scholar 

  5. Huang AC, Jiang T, Liu YX, Bai YC, Reed J, Qu B, Goossens A, Nutzmann HW, Bai Y. Osbourn A A specialized metabolic network selectively modulates Arabidopsis root microbiota. Science. 2019;364(6440):546–6.

    Google Scholar 

  6. Kessler A, Baldwin IT. Defensive function of herbivore-induced plant volatile emissions in nature. Science. 2001;291(5511):2141–4.

    CAS  PubMed  Google Scholar 

  7. Lee C, Kim T, Seung N-PR. Rhee genomic signatures of specialized metabolism in plants. Science. 2014;344(6183):510–3.

    Google Scholar 

  8. Aharoni A, Jongsma MA, Bouwmeester HJ. Volatile science? Metabolic engineering of terpenoids in plants. Trends Plant Sci. 2005;10(12):594–602.

    CAS  PubMed  Google Scholar 

  9. Sawamura M. Volatile components of essential oils of the Citrus genus. Recent Resdevelagricultural & Food Chem. 2000;4(1):131–64.

    CAS  Google Scholar 

  10. Vekiari SA, Protopapadakis EE, Papadopoulou P, Papanicolaou D, Panou C, Vamvakias M. Composition and seasonal variation of the essential oil from leaves and peel of a cretan lemon variety. J Agric Food Chem. 2002;50(1):147–53.

    CAS  PubMed  Google Scholar 

  11. Zhang M, Su P, Zhou YJ, Wang XJ, Zhao YJ, Liu YJ, Tong YR, Hu TY, Huang LQ, Gao W. Identification of geranylgeranyl diphosphate synthase genes from Tripterygium wilfordii. Plant Cell Rep. 2015;34(12):2179–88.

    CAS  PubMed  Google Scholar 

  12. Cao R, Zhang Y, Mann FM, Huang C, Mukkamala D, Hudock MP, Mead ME, Prisic S, Wang K, Lin FY, et al. Diterpene cyclases and the nature of the isoprene fold. Proteins. 2010;78(11):2417–32.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Herde M, Gartner K, Kollner TG, Fode B, Boland W, Gershenzon J, Gatz C, Tholl D. Identification and regulation of TPS04/GES, an Arabidopsis geranyllinalool synthase catalyzing the first step in the formation of the insect-induced volatile C16-homoterpene TMTT. Plant cell. 2008;20(4):1152–68.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Hilker M, Fatouros NE. Plant responses to insect egg deposition. Annu Rev Entomol. 2015;60:493–515.

    CAS  PubMed  Google Scholar 

  15. Balkema-Boomstra AG, Zijlstra S, Verstappen FW, Inggamer H, Mercke PE, Jongsma MA, Bouwmeester HJ. Role of cucurbitacin C in resistance to spider mite (Tetranychus urticae) in cucumber (Cucumis sativus L). J Chem Ecol. 2003;29(1):225–35.

    CAS  PubMed  Google Scholar 

  16. Bohlmann J, Martin D, Oldham NJ, Gershenzon J. Terpenoid secondary metabolism in Arabidopsis thaliana: cDNA cloning, characterization, and functional expression of a myrcene/(E)-beta-ocimene synthase. Arch Biochem Biophys. 2000;375(2):261–9.

    CAS  PubMed  Google Scholar 

  17. Nagegowda DA. Plant volatile terpenoid metabolism: biosynthetic genes, transcriptional regulation and subcellular compartmentation. FEBS Lett. 2010;584(14):2965–73.

    CAS  PubMed  Google Scholar 

  18. Lichtenthaler HK. The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Ann Rev Plant Physiol Plant Mol Biol. 1999;50(1):47–65.

    CAS  Google Scholar 

  19. Sapir-Mir M, Mett A, Belausov E, Tal-Meshulam S, Frydman A, Gidoni D, Eyal Y. Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes. Plant Physiol. 2008;148(3):1219–28.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Chen F, Tholl D, Bohlmann J, Pichersky E. The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 2011;66(1):212–29.

    CAS  PubMed  Google Scholar 

  21. He J, Bouwmeester HJ, Dicke M, Kappers IF. Genome-wide analysis reveals transcription factors regulated by spider-mite feeding in Cucumber (Cucumis sativus). Plants (Basel). 2020;9(8):1014.

    CAS  PubMed  Google Scholar 

  22. Martin DM, Aubourg S, Schouwey MB, Daviet L, Schalk M, Toub O, Lund ST, Bohlmann J. Functional annotation, genome organization and phylogeny of the grapevine (Vitis vinifera) terpene synthase gene family based on genome assembly, FLcDNA cloning, and enzyme assays. BMC Plant Biol. 2010;10:226.

    PubMed  PubMed Central  Google Scholar 

  23. Dornelas MC. Mazzafera P A genomic approach to characterization of the Citrus terpene synthase gene family. Genet Mol Biol. 2007;30(3):832–40.

    CAS  Google Scholar 

  24. Alquezar B, Rodriguez A, de la Pena M, Pena L. Genomic analysis of terpene synthase family and functional characterization of seven sesquiterpene synthases from Citrus sinensis. Front Plant Sci. 2017;8:1481.

    PubMed  PubMed Central  Google Scholar 

  25. Xu Y, Zhu C, Xu C, Sun J, Grierson D, Zhang B, Chen K. Integration of metabolite profiling and transcriptome analysis reveals genes related to volatile terpenoid metabolism in Finger Citron (C. medica var. sarcodactylis). Molecules. 2019;24(14):2564.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Arimura G, Huber DP, Bohlmann J. Forest tent caterpillars (Malacosoma disstria) induce local and systemic diurnal emissions of terpenoid volatiles in hybrid poplar (Populus trichocarpa x deltoides): cDNA cloning, functional characterization, and patterns of gene expression of (-)-germacrene D synthase, PtdTPS1. Plant J. 2004;37(4):603–16.

    CAS  PubMed  Google Scholar 

  27. De Vos M, Van Oosten VR, Van Poecke RM, Van Pelt JA, Pozo MJ, Mueller MJ, Buchala AJ, Metraux JP, Van Loon LC, Dicke M, et al. Signal signature and transcriptome changes of Arabidopsis during pathogen and insect attack. Mol Plant Microbe Interact. 2005;18(9):923–37.

    CAS  PubMed  Google Scholar 

  28. Kant MR, Ament K, Sabelis MW, Haring MA, Schuurink RC. Differential timing of spider mite-induced direct and indirect defenses in tomato plants. Plant Physiol. 2004;135(1):483–95.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Martel C, Zhurov V, Navarro M, Martinez M, Cazaux M, Auger P, Migeon A, Santamaria ME, Wybouw N, Diaz I, et al. Tomato whole genome transcriptional response to Tetranychus urticae identifies divergence of spider mite-induced responses between tomato and Arabidopsis. Mol Plant Microbe Interact. 2015;28(3):343–61.

    CAS  PubMed  Google Scholar 

  30. Schnee C, Kollner TG, Gershenzon J, Degenhardt J. The maize gene terpene synthase 1 encodes a sesquiterpene synthase catalyzing the formation of (E)-beta-farnesene, (E)-nerolidol, and (E,E)-farnesol after herbivore damage. Plant Physiol. 2002;130(4):2049–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhurov V, Navarro M, Bruinsma KA, Arbona V, Santamaria ME, Cazaux M, Wybouw N, Osborne EJ, Ens C, Rioja C, et al. Reciprocal responses in the interaction between Arabidopsis and the cell-content-feeding chelicerate herbivore spider mite. Plant Physiol. 2014;164(1):384–99.

    CAS  PubMed  Google Scholar 

  32. He J, Verstappen F, Jiao A, Dicke M, Bouwmeester HJ. Kappers IF terpene synthases in cucumber (Cucumis sativus) and their contribution to herbivore-induced volatile terpenoid emission. New Phytol. 2022;233(2):862–77.

    CAS  PubMed  Google Scholar 

  33. Vom Endt D, Soares e Silva M, Kijne JW, Pasquali G, Memelink J. Identification of a bipartite jasmonate-responsive promoter element in the Catharanthus roseus ORCA3 transcription factor gene that interacts specifically with AT-Hook DNA-binding proteins. Plant Physiol. 2007;144(3):1680–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Raffaele S, Rivas S. Regulate and be regulated: integration of defense and other signals by the AtMYB30 transcription factor. Front Plant Sci. 2013;4:98.

    PubMed  PubMed Central  Google Scholar 

  35. Xu XP, Chen CH, Fan BF, Chen ZX. Physical and functional interactions between pathogen-induced Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors. Plant Cell. 2006;18(5):1310–26.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Knudsen JT, Eriksson R, Gershenzon J, Stahl B. Diversity and distribution of floral scent. Bot Rev. 2006;72(1):1–120.

    Google Scholar 

  37. Boachon B, Junker RR, Miesch L, Bassard JE, Hofer R, Caillieaudeaux R, Seidel DE, Lesot A, Heinrich C, Ginglinger JF, et al. CYP76C1 (cytochrome P450)-mediated linalool metabolism and the formation of volatile and soluble linalool oxides in Arabidopsis flowers: a strategy for defense against floral antagonists. Plant Cell. 2015;27(10):2972–90.

    CAS  PubMed  PubMed Central  Google Scholar 

  38. He J, Fandino RA, Halitschke R, Luck K, Kollner TG, Murdock MH, Ray R, Gase K, Knaden M, Baldwin IT, et al. An unbiased approach elucidates variation in (S)-(+)-linalool, a context-specific mediator of a tri-trophic interaction in wild tobacco. Proc Natl Acad Sci U S A. 2019;116(29):14651–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Van Schie CC, Haring MA. Schuurink RC Tomato linalool synthase is induced in trichomes by jasmonic acid. Plant Mol Biol. 2007;64(3):251–63.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Rudmann AA, Aldrich JR. Chirality determinations for a tertiary alcohol - ratios of linalool enantiomers in insects and plants. J Chromatogr. 1987;407:324–9.

    CAS  Google Scholar 

  41. Giglio A, Brandmayr P, Dalpozzo R, Sindona G, Tagarelli A, Talarico F, Brandmayr TZ. Ferrero EA the defensive secretion of Carabus lefebvrei Dejean 1826 pupa (Coleoptera, Carabidae): gland ultrastructure and chemical identification. Microsc Res Tech. 2009;72(5):351–61.

    CAS  PubMed  Google Scholar 

  42. Maczka W, Duda-Madej A, Grabarczyk M, Winska K. Natural compounds in the battle against microorganisms-linalool. Molecules. 2022;27(20):6928.

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Raguso RA. More lessons from linalool: insights gained from a ubiquitous floral volatile. Curr Opin Plant Biol. 2016;32:31–6.

    CAS  PubMed  Google Scholar 

  44. Zhang HP, Chen MJ, Wen H, Wang ZH, Chen JJ, Fang L, Zhang HY, Xie ZZ, Jiang D, Cheng YJ, et al. Transcriptomic and metabolomic analyses provide insight into the volatile compounds of citrus leaves and flowers. BMC Plant Biol. 2020;20:7.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Shimada T, Endo T, Fujii H, Rodriguez A, Pena L, Omura M. Characterization of three linalool synthase genes from Citrus unshiu Marc. And analysis of linalool-mediated resistance against Xanthomonas citri subsp. citri and Penicilium italicum in citrus leaves and fruits. Plant Sci. 2014;229:154–66.

    CAS  PubMed  Google Scholar 

  46. Shimada T, Endo T, Rodríguez A, Fujii H, Goto S, Matsuura T, Hojo Y, Ikeda Y, Mori IC. Fujikawa T ectopic accumulation of linalool confers resistance to Xanthomonas citri subsp. citri in transgenic sweet orange plants. Tree Physiol 2017(5):654.

  47. Dorman H, Deans SG. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J Aappl Microbiol. 2010;88(2):308–16.

    Google Scholar 

  48. Park SN, Lim YK, Freire MO, Cho E, Jin D. Kook JK Antimicrobial effect of linalool and alpha-terpineol against periodontopathic and cariogenic bacteria. Anaerobe. 2012;18(3):369–72.

    CAS  PubMed  Google Scholar 

  49. Yamasaki Y, Kunoh H, Yamamoto H, Akimitsu K. Biological roles of monoterpene volatiles derived from rough lemon (Citrus jambhiri Lush) in citrus defense. J Gen Plant Pathol. 2007;73(3):168–79.

    CAS  Google Scholar 

  50. Droby S, Eick A, Macarisin D, Cohen L, Rafael G, Stange R, McColum G, Dudai N, Nasser A, Wisniewski M, et al. Role of citrus volatiles in host recognition, germination and growth of Penicillium digitatum and Penicillium italicum. Postharvest Biol Tec. 2008;49(3):386–96.

    CAS  Google Scholar 

  51. Alves S, Duarte A, Sousa S, Domingues FC. Study of the major essential oil compounds of Coriandrum sativum against Acinetobacter baumannii and the effect of linalool on adhesion, biofilms and quorum sensing. Biofouling. 2016;32(2):155–65.

    CAS  PubMed  Google Scholar 

  52. Shimada T, Endo T, Fujii H, Rodriguez A, Yoshioka T, Pena L, Omura M. Biological and molecular characterization of linalool-mediated field resistance against Xanthomonas citri subsp. citri in citrus trees. Tree Physiol. 2021;41(11):2171–88.

    CAS  PubMed  Google Scholar 

  53. De Carvalho SA, de Carvalho Nunes WM, Belasque J Jr, Machado MA, Croce-Filho J, Bock CH. Abdo Z comparison of resistance to asiatic citrus canker among different genotypes of citrus in a long-term canker-resistance field screening experiment in Brazil. Plant Dis. 2015;99(2):207–18.

    PubMed  Google Scholar 

  54. Matsumura M, Nomoto M, Itaya T, Aratani Y, Iwamoto M, Matsuura T, Hayashi Y, Mori T, Skelly MJ. Yamamoto YY Mechanosensory trichome cells evoke a mechanical stimuli–induced immune response in Arabidopsis thaliana. Nat Commun. 2022;13(1):1216.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Ahmad MM, Rehman SU, Anjum FM, Bajwa EE. Comparative physical examination of various citrus peel essential oils. Int J Agric Biol. 2006;8(2):186–90.

    Google Scholar 

  56. Uji Y, Ozawa R, Shishido H, Taniguchi S, Takabayashi J, Akimitsu K, Gomi K. Isolation of a sesquiterpene synthase expressing in specialized epithelial cells surrounding the secretory cavities in rough lemon (Citrus jambhiri). J Plant Physiol. 2015;180:67–71.

    CAS  PubMed  Google Scholar 

  57. Miller B, Madilao LL, Ralph S, Bohlmann J. Insect-induced conifer defense. White pine weevil and methyl jasmonate induce traumatic resinosis, de novo formed volatile emissions, and accumulation of terpenoid synthase and putative octadecanoid pathway transcripts in Sitka spruce. Plant Physiol. 2005;137(1):369–82.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Zulak KG, Lippert DN, Kuzyk MA, Domanski D, Chou T, Borchers CH, Bohlmann J. Targeted proteomics using selected reaction monitoring reveals the induction of specific terpene synthases in a multi-level study of methyl jasmonate-treated Norway spruce (Picea abies). Plant J. 2009;60(6):1015–30.

    CAS  PubMed  Google Scholar 

  59. Fang XF, Meng XN, Zhang J, Xia MH, Cao SQ, Tang XF, Fan TT. AtWRKY1 negatively regulates the response of Arabidopsis thaliana to Pst DC3000. Plant Physiol Bioch. 2021;166(1):799–806.

    CAS  Google Scholar 

  60. Logemann E, Birkenbihl RP, Ülker B. Somssich IE An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods. 2006;2:16.

    PubMed  PubMed Central  Google Scholar 

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We sincerely thank Prof. Fu Xingzheng for providing us with vectors to help us complete the experiment, and Profs. Ian T. Baldwin and Xiaochun Zhao for editing the final draft of the manuscript.


This work was funded by the National Key Research and Development Program of China (2021YFD1400800), the Fundamental Research Funds for the Central Universities (SWU120067), the Venture & Innovation Support Program for Chongqing Overseas Returnees (7820100514), and Innovation Research 2035 Pilot Plan of Southwest University (SWU-5331000008).

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Q.W., X.W. and J.H. conceptualized the project; X.W. and J.H. administrated the project and supervised the research, and acquired the funding and resources; Q.W. X.W. L.R. and H.Y. did investigation; Q.W. wrote the original draft, which C.Z., X.W. and J.H. reviewed & edited: All authors read and approved the final manuscript.

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Correspondence to Xuefeng Wang or Jun He.

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Wang, Q., Wang, X., Huang, L. et al. Promoter characterization of a citrus linalool synthase gene mediating interspecific variation in resistance to a bacterial pathogen. BMC Plant Biol 23, 405 (2023).

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