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

Background 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. Results 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. Conclusions 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. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-023-04413-6.


Background
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 Gramnegative 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.
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).
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.
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.
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).
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).

Discussion
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 cankerresistance 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.

Conclusions
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.

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 (OD 600 = 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 (OD 600 = 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 OD 600 = 0.5.The culture was centrifuged at 5000 g for 5 min, and resuspended in sterile 10 mM MgCl 2 buffer to OD 600 = 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 MgCl 2 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 (http://citrus.hzau.edu.cn/download.php).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).
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 (http://bioinformatics.psb.ugent.be/webtools/plantcare/)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 T 3 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.

Fig. 1
Fig. 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)

Fig. 2
Fig. 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

Fig. 4 Fig. 3
Fig. 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

Fig. 5
Fig. 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