Floral scent components in Rhododendron fortunei and its regulation by gene expression of S-adenosyl-l-methionine:benzoic acid carboxyl methyl transferase (BAMT)

Background: Rhododendron fortunei belongs to a scented Rhododendron species native to China, which produces fragrant owers of great ornamental and environmental values. However, the scents in R. fortunei have not yet been investigated. Results: The results showed that three main VOCs measured from highest to lowest are methyl benzoates, terpenes and fatty acid derivatives. Their content increased after the ower bud opening and reached the highest at half to full blossom. In a ower most VOC contents were measured in petals and only trace amount in other tissues such as stamen, pistil. A small amount of VOCs was determined in leaves as well. All aromatic values were almost corresponded to the contents of three main VOCs, indicating that the ower fragrance arises truly from these VOC components. To understand the mechanism of the formation of this main type fragrance and its regulation, we rstly isolate a gene of RfBAMT from petal of R. fortunei by using homologous cloning and RACE technology. The full length of its cDNA was 1383 bp (cid:0) with an open reading frame of 1104 bp, encoding a total of 368 amino acids. The phylogenetic tree analysis showed that RfBAMT was the closest to the BSMT of Camellia japonica, belonging to methyltransferases family. Then we measured the expression level of RfBAMT again at four ower developmental stages and in different ower tissues and leaves. The results showed that the expression level of this gene was highly positively correlated with the emitted content of methyl benzoates in the owering, implying that RfBAMT plays a pivotal role in the formation and regulation of methyl benzoates in R. fortunei . Conclusions: This research showed that the RfBAMT was cloned and identied in our study and its expression level was highly positively correlated with the emitted content of methyl benzoates in the owers and leaves, which indicated this gene may play an important role on regulation of methyl benzoate synthesis in R. fortunei .


Background
Rhododendron is the largest genus in the family Ericaceae, with as many as 1024 species and amongst them 567 species representing 6 subgenera are native to China [1][2][3]. They are very popular grown as one of the major horticultural plants and mostly used for landscaping or indoor beauti cation. There is a plethora of varieties with different ower colors or shapes of Chinese Rhododendron but only a few wild species called the fragrant Rhododendron produce owers of fragrance and habitat predominately in mountainous areas. The fragrant Rhododendron is obviously welcome in the ower plant market due to their strong aesthetic and emotional values. Rhododendron fortunei belongs to a fragrant Rhododendron species, which produces fragrant owers. However, it is di cult to be popularized for commercial purposes because of its short owering period and di culties in adaption to grow in low altitudes. It is plausible to breed fragrant Rhododendron by transferring the scent traits of R. fortunei to non-scent species through an approach of genetic engineering [4].
To explore the genetic material of R. fortunei for transforming oral scent in other Rhododendron species, we investigate the volatile constituents and the mechanism of it formation and regulation in R. fortunei. We rstly determined the scent volatile constituents in this species by headspace solid-phase micro-extraction combined with gas chromatography-mass spectrometry. The volatile components and relative contents in R. fortunei at four different owering stages and in different tissues were measured and results shown that the main VOC constituent is benzenoid class compounds. As it is known that S-adenosyl-l-methionine: benzoic acid carboxyl methyl transferase (BAMT) catalyzes the nal step to form methyl benzoates [5], we then cloned the BAMT gene from this species and study its expression pattern in relation to the accumulation of aroma compositions. We hope that our experiments could provide some data to explain the mechanism of the aroma formation and regulation in R. fortunei and eventually identify the gene targets for molecular breeding of fragrant Rododendron cultivars.

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VOC constituents and related aromatic values of compounds emitted from the owers of R. fortunei Eighteen VOCs were detected in R. fortunei, which can be divided into three classes: benzenoids, terpenoids, and fatty acid derivatives (Table 1 and Fig. 1b). The content of benzenoids increased after the ower bud opening and reached the highest at full blossom from 0.23 to 12.79 mg/kg·FW; while terpenoids and fatty acid derivatives increased and reached the highest at half blossom from 0.21 to 5.53 mg/kg·FW, and for from 0.55 to 4.72 mg/kg·FW, respectively (Fig. 1b).
Among them 4 kinds of benzenoids derived from the shikimic acid pathway accounted for the highest content of VOC in each sample, which indicated that the benzenoids were the most dominant VOC components in oral scent of R. fortunei. A total of 5 terpenoids derived from the MEP and MVA pathways were also detected in this study. Terpenoids are the secondary abundant metabolites commonly found in plants [6], most of which have a strong sweet, oral and woody aroma. Fatty acid derivatives are the most diverse compounds having 15 species detected including alkanes, alkenes, alcohols, aldehydes and other compounds, derived from the lipoxygenase pathway [7]. However, contents of fatty acid derivatives were signi cantly lower than both the contents of benzenoids and terpenoids. The VOCs in different tissues and leaves were also measured and showed that most amount of VOCs were released from the petal and trace amount from stamen, pistil and even from leaves. The total release of VOCs from ower petal were 5.89-fold, 15.73-fold and 6.6-fold higher than from stamen, pistil and leaf, respectively ( Fig. 2b and Table 2). Again the compositions from high to low in all tissues and leaves was benzenoids the highest, followed by the terpenoids and then the fatty acid derivatives. The aromatic value or odor unit of a speci c released compounds were calculated based on the content of the VOCs dividing by its respective odor threshold [8], which re ects the degree of scent olfactory perception by human. If the odor unit is more than 1, we regard it as a characteristic aroma component [9]. Since benzaldehyde and ethyl benzoate has a high aroma threshold, the odor unit is less than 1, so it is not a characteristic aroma component. According to this criteria the characteristic aroma components in owers at different developmental stages and tissues of R. fortunei were listed in Table 1 and 3. Moreover, the aromatic value of each VOCs was calculated and shown in Table 2 and 4; Fig. 1c and 2c. The data shows that all aromatic values were almost corresponded to the contents of three main VOCs, indicating the ower fragrant odor arisen truly from these VOC components.  We learned from above analysis that benzenoids are the main oral scent in R. fortune. It is also known that S-adenosyl-lmethionine: benzoic acid carboxyl methyl transferase (BAMT) catalyzes the nal step to form methyl benzoates shown in Fig. 3a [5]. To understand the function of this enzyme in the formation of benenoids in the owers of R. fortune, we rst clone this gene. The petal cDNA of R. fortunei was used as a template for RT-PCR ampli cation, and obtained the middle segment sequence by cloning. And then the 3' and 5' ends fragments were cloned by RACE technology respectively. The conserved region was 806 bp, the 3' ends was 788 bp, and the 5' ends was 614 bp (Fig. 3b). These three sequences were spliced to obtain a sequence of 1383 bp which was named RfBAMT (Fig. 3c). RfBAMT contains an ATG start codon and a TGA stop codon, a tail signal AATAAA and a 26 bp polyA tail. Furthermore, the gene contains an ORF of 1104 bp, and an untranslated region with 5' UTR of 12 bp and 3'UTR of 267 bp. The deduced protein of the coding region containing 368 amino acid residues (Fig. 3c), whose theoretical molecular weight is 41.09 kDa with an isoelectric point 5.19 predicted by ProtParam. Conserved domains Research tool showed that the position of 38-366 amino acids contained a Methyltransf-7 superfamily's conserved region which is the characteristic domain of methyltransferases, therefore identi ed as a true BAMT in Rhododendron.

Expression pattern of RfBAMT
To clarify the role of RfBAMT on regulation of benezoid formation, we measured the expression of RfBAMT gene at four owering stages and in different ower tissues in comparison to in leaves of R. fortunei. The results showed that the expression increased rst from the ower buds stage and reached the peak of 3.48 fold increase at the full opening stage and then decreased after the owers started to wilt (Fig. 5a). RfBAMT was widely expressed in these tissues and leaves. However, the expression level in the petal was signi cantly 16.67 fold 1.64 fold and 1.59 higher than that in stamen, pistil and leaf, respectively (Fig. 5b). Together with the results on the measurements of oral scent contents (Fig. 1b and Fig. 2b), it was revealed that this gene expression was highly correlated to the content of oral VOCs in the oral tissues and at different owering stages, suggesting RfBAMT function in the regulation of benezoid metabolism in R. fortune.

Discussion
Determination of oral compositions in R. fortunei Floral scent is an important component part of plant volatile compounds [10], which is a complex mixture of many lowmolecular-mass and volatile compounds [11]. So far, more than 1,700 oral volatile organic compounds (VOCs) have been identi ed from 90 different families of plants, most of which belong to terpenoids, benzenoids, and fatty acid derivatives [7,12,13].
In this study, three classes of the oral volatile compounds were detected in R. fortunei and among them more than 55% of VOC were benzenoids and thus it was the dominant scent constituent in the released compounds of owers at the owering stages and different tissues ( Fig. 1b and Fig. 2b). Benzenoids are commonly found in plant VOC [14] such as in Prunus mume [15], European Narcissus [9] and especially in rose [16] that more than 50% of the total released VOC is benenoids. Therefore, our results are consistent with these above reports. However, Su et all [17] found that the relative content of benzenoids in Rhododendron was only 16.6% of the total VOC released from the owers which was lower than we measured in R. fortunei. This discrepancy could be caused by many factors such as plant material, cultivation environment and analysis conditions [18]. Methyl benzoate belongs to benzenoids and full of strong wintergreen and eucalyptus oil scent which can be used to formulate rose avor. In addition, it is also used as an additive for cosmetics and foods [19]. As a benzenoid, methyl benzoate has a rich aroma of wintergreen and eucalyptus oil scent, and is the main aroma component of owers [20]. Verdonk et all [21] found that the release of methyl benzoate was large in Petunia; the oral composition analysis of Snapdragon indicated that the relative content of methyl benzoate was as high as 60% [5]; Zhang et all [22] found that the release amount of methyl benzoate in scented Lilium was higher, but it could not be detected in light-scented Lilium. This study demonstrated that the content of methyl benzoate was high in the owering stage and different tissues in R. fortunei, and the aroma threshold was also low (0.028 mg/kg). It can be inferred that methyl benzoate identi ed as a key aroma component of R. fortunei.
Terpenoids and fatty acid derivates were also abundantly detected in the R. fortunei owers, accounting for 10.43% and 8.69% of the total VOC emitted from the full opening owers (Fig. 1b and Fig. 2b ). As we mentioned above, these 3 classes of compounds contribute to the aroma value as well ( Fig. 1c and Fig. 2c), which demonstrates the fragrance comes indeed from the combined three classes of VOCs.
Regulation of oral scent formation by RfBAMT in R. fortunei As above shown that benzenoids are the dominant scent compounds in R. fortunei, we then focused on the biochemistry mechanism of its synthesis. Methyl benzoate is the main compound in benzenoid class and it is synthesized by benzoic acid/salicylic acid carboxyl methyltransferase, catalyzing the transfer of the methyl donor benzoic acid to corresponding acids (Fig. 3a) [5]. The BAMT has been isolated from plants such as Nicotiana suaveolens [23], Petunia hybrid [23] and Snapdragon [5]. In this experiment, the full-length cDNA sequence of BAMT in R. fortunei was isolated by homologous and RACE cloning technology and identi ed as RfBAMT which could code putatively the enzyme of BAMT in R. fortunei (Fig. 4).
Then we examined its gene expression patterns both in different owering stages and in different oral tissues and leaves of R. fortunei and our results showed that the expression level (Fig. 5) was highly corresponded to the content of methyl benzoate (MeBA) (Fig. 1b and Fig. 2b), implying RfBAMT function in regulation of MeBA biosynthesis. However, to prove this function, we think that it is still required to measure the enzyme activity or protein amount of BAMT translated from its transcripts by biochemical assay or Western blot. Then we can make transgenic plants by knocking out or overexpressing the RfBAMT gene to assess its distinct role in the metabolism of benzenoids in R. fortunei and clarify whether the regulation of benzenoid biosynthesis is precursor-regulated when this enzyme only partial contributes to the total amount of MeBA content [16]. Furthermore, the transcriptomic approach could be used to address the oral scent mechanism in Rhododendron by comparing the scented R. fortunei with the non-scented R. hybridae, in hope that any transcription factor could be found, like Myb transcription factor ODORANT1 in petunia [24].

Conclusion
In summary, methyl benzoate was the dominant scent components emitted from the owers of R. fortunei. At present, only the RfBAMT was cloned and identi ed in our study and its expression level was highly positively correlated with the emitted content of methyl benzoates in the owers and leaves, which indicated this gene may play an important role on regulation of methyl benzoate synthesis in R. fortunei. To understand further the molecular mechanism of the regulation of the oral scent synthesis in R. fortunei, studies on other key genes involved in the biosynthesis of other main VOC components such as terpenoids and fatty acid derivatives are evidently warranted to illustrate the regulation network of oral scent metabolism and eventually identify the genetic targets for breeding fragrant varieties of Rhododendron.

Materials Plant Material
The fresh ower petal during bud stage, middle opening stage, full opening stage and wilting stage (Fig. 1a) and the ower petal, stamen, and pistil at full opening stage and leaves (Fig. 2a) of R. fortunei were collected from Siming Mountain National Forest Park in Ningbo, China. Parts of them were directly used to determinate VOCs, and the remained parts were placed at -80℃ for storage. Wenguang Hu undertook the formal identi cation of the samples and provide details of specimens deposite in Flora of China. All R. fortunei material was obtained with permission.

Floral scent collection and analysis
The HS-SPME-GC-MS was used to collect the oral scent. Aging the 65µmol/L PDMS/DVB SPME bers at injection port of GC and set the temperature at 250℃. Weigh 5g of the shredded sample rapidly and put it into an 8mL headspace bottle. And then insert the SPME injector into a sealed bottle. Headspace extraction for 1h at 30℃ water bath with 500 r/min stirring. After the extraction, insert the SPME bers into injection port of GC and resolve for 5 min immediately. Three biological replicate measurements were performed on each sample.
Chromatographic conditions: Agilent lichrosorb 19091S-433 (30m x 250μm x 0.25μm); the temperature of column is 40℃; the injector was operated splitless at a temperature of 250℃ with He as a carrier gas at 1.0mL/min. The following temperature program was used: initial temperature of 40℃ (2min hold), increase to 160℃ at 3℃/min, 10℃/min ramp to 200℃, followed by a 20℃/min ramp to 300℃ (3min hold), with the port in splitless injection mode 5 .
Preliminary identi cation of the VOCs was made by searching the NIST library and checked according to its retention index. The Identi cation results with a matching degree above 80 (maximum 100) are used [25]. Benzyl benzoate (0.186g/L) was used as an internal standard to quantify the volatile compounds of the oral scent. x10 -3 / sample weight (kg). Then the aromatic value was calculated for each components respectively according to the threshold of odor [6].
Isolation of the full-length cDNA Total RNA was isolated from R. fortunei petal using the RNAprep Pure Plant Kit (TIANGEN, China). After passing the NanoDropTM2000 and 1% agarose gel electrophoresis, it was reverse transcribed to rst-strand cDNA (CWBIO, China). Download the full length sequence of BAMT gene from plants similar to Rhododendron in GenBank, and then using ClustalW to nd their conserved sequence. Finally, a pair of degenerate primers BAMT-F1 and BAMT-R1 were designed for PCR ampli cation by Primer 5.0 ( Table 5). The PCR reaction procedure was determined as follows: predenaturation at 94°C for 5 min, denaturation at 94°C for 30s, annealing at 50°C for 30s, extension at 68°C for 1 min, a total of 35 cycles, and nally, 72°C. Extend for 10 min and store at 4°C (TRANS, China).
The PCR product was detected by 1.0% agarose gel electrophoresis. The fragment was extracted with plastic recycling kit, and then cloned to the vector pEASY-Blunt Zero (TRANS, China) and sequenced by Shanghai Sangon Biotech Company.
Gene-speci c primers (5'GSP and 3'GSP, Table 5) were designed based on the sequence of middle segment. The SMARTer® RACE 5'/3'Kit (Takara) was used to isolate the 3' and 5' ends of the cDNA, and spliced a full-length cDNA sequence by DNAMAN.

Bioinformatics analysis
The open reading frame of nucleotide sequence and deduced amino acid sequence was analysed by ORF nder tool on the http://www.NCBI.com website. Conserved domains Research tool was used to predictive gene domain. Molecular weight and isoelectric point of the protein were predicted by the online software ProtParam (http://web.expasy.org/protparam/) on ExPASy. Homology evolution of amino acid sequence and members of the gene family were analysed by DNAMAN software, and constructed phylogenetic tree by MEGA 6.06 software.

Quantitative real-time PCR
To perform quantitative real-time PCR (qRT-PCR) analysis, total RNA was isolated from different ower developing stages and oral parts of R. fortunei. Primers for qRT-PCR were designed based on the RfBAMT cDNA sequence (Table1). EF1α was selected as internal reference gene for each sample (Table 5). qRT-PCR was carried out using the Bio-Rad real-time PCR system with ChamQ™SYBR®Color qPCR Master Mix. The program was: 95℃ degeneration 3 sec, 40 cycles of 95℃ for 10 s, 60℃ for 30s, uorescent signal acquisition in 60℃. All the qRT-PCR results were presented as means ± SD of three biological replicates. The relative expression level of genes were calculated by the 2 -Δ Δ Ct equation.

Declarations
Ethics approval and consent to participate Not applicable.

Consent to publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This research was supported by major Public Welfare Agriculture Project in Ningbo (2014C11002). We thank the foundation for economic support. The funding organization provided the nancial support to the research projects, and involved in the design of the study. Authors'contributions