Next Generation Sequencing Unravels the Biosynthetic Ability of Spearmint (Mentha Spicata) Peltate Glandular Trichomes Through Comparative Transcriptomics

Next generation sequencing unravels the biosynthetic ability of Spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics

Jin et al.

Jin et al. BMC Plant Biology 2014, 14:292 http://www.biomedcentral.com/1471-2229/14/292 Jin et al. BMC Plant Biology 2014, 14:292 http://www.biomedcentral.com/1471-2229/14/292

RESEARCH ARTICLE Open Access Next generation sequencing unravels the biosynthetic ability of Spearmint (Mentha spicata) peltate glandular trichomes through comparative transcriptomics Jingjing Jin1,2,3, Deepa Panicker1, Qian Wang1, Mi Jung Kim1, Jun Liu3, Jun-Lin Yin1, Limsoon Wong2, In-Cheol Jang1,4, Nam-Hai Chua3 and Rajani Sarojam1*

Abstract Background: Plant glandular trichomes are chemical factories with specialized metabolic capabilities to produce diverse compounds. Aromatic mint plants produce valuable essential oil in specialised glandular trichomes known as peltate glandular trichomes (PGT). Here, we performed next generation transcriptome sequencing of different tissues of Mentha spicata (spearmint) to identify differentially expressed transcripts specific to PGT. Our results provide a comprehensive overview of PGT’s dynamic metabolic activities which will help towards pathway engineering. Results: Spearmint RNAs from 3 different tissues: PGT, leaf and leaf stripped of PGTs (leaf-PGT) were sequenced by Illumina paired end sequencing. The sequences were assembled de novo into 40,587 non-redundant unigenes; spanning a total of 101 Mb. Functions could be assigned to 27,025 (67%) unigenes and among these 3,919 unigenes were differentially expressed in PGT relative to leaf - PGT. Lack of photosynthetic transcripts in PGT transcriptome indicated the high levels of purity of isolated PGT, as mint PGT are non-photosynthetic. A significant number of these unigenes remained unannotated or encoded hypothetical proteins. We found 16 terpene synthases (TPS), 18 cytochrome P450s, 5 lipid transfer proteins and several transcription factors that were preferentially expressed in PGT. Among the 16 TPSs, two were characterized biochemically and found to be sesquiterpene synthases. Conclusions: The extensive transcriptome data set renders a complete description of genes differentially expressed in spearmint PGT. This will facilitate the metabolic engineering of mint terpene pathway to increase yield and also enable the development of strategies for sustainable production of novel or altered valuable compounds in mint. Keywords: Spearmint, Next generation sequencing, Transcriptome, Glandular trichomes, Terpenes, Carvone, Terpene synthases

Background These terpenes provide protection for plants against a Plants produce an enormous variety of specialised me- variety of herbivores and pathogens [3] and are also tabolites among which terpenes are the largest and most commercially quite valuable. Therefore, the processes by structurally diverse class of natural products. They are which they are synthesised and stored in plants are main the main components of plant essential oils. Many of target for genetic manipulation for increased yield. But these terpenes are produced and stored in specialised our knowledge about the development of secretory glan- secretory structures called glandular trichomes [1,2]. dular trichomes and terpene production and its regulation is very limited making it difficult to engineer these metabolic pathways [4,5]. * Correspondence: [email protected] 1Temasek Life Sciences Laboratory, 1 Research Link, National University of Aromatic essential oil produced by Mentha species is Singapore, Singapore 117604, Singapore the source of the best known monoterpenes, menthol Full list of author information is available at the end of the article

© 2014 Jin et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jin et al. BMC Plant Biology 2014, 14:292 Page 2 of 14 http://www.biomedcentral.com/1471-2229/14/292

and carvone, which form the principal components of space that is formed by the separation of the cuticle from mint oil. They are extensively used in flavour and fra- the apical cells and the essential oil is secreted into this grance industries, pharmaceuticals and cosmetic products cavity [12] (Figure 1B). It is known that new glands keep [6]. Peppermint variety mostly produces menthol whereas initiating on the leaf till expansion ceases and the mono- in spearmint variety carvone dominates [7,8]. From the terpene content and compositions change with the age PGT of peppermint variety ( Mentha X piperita), 1,316 of the leaf [13-16]. Different studies have indicated that randomly selected cDNA clones, or expressed sequence monoterpene biosynthesis is most active in young 12–20 tags (ESTs) were produced, which led to the identification day old leaves of peppermint after which the rate of syn- of many genes, enzymes and substrates involved in the thesis slowly declines [17-19]. We performed gas chroma- main menthol essential oil biosynthetic pathway [9,10]. tography–mass spectrometry (GC-MS) analysis on young Given the technical limitations at their time of study, an spearmint leaves (about 1–2cminlength)andfound EST approach would possibly identify only cDNAs which abundance of both limonene and carvone monoterpenes are abundant in PGT. A recent proteomic analysis of (Figure 2). Limonene is the first committed step towards spearmint PGT identified 1,666 proteins of which 57 were carvone pathway. In addition to these monoterpenes, the predicted to be involved in secondary metabolism [11]. presence of sesquiterpenes was also observed. This indi- But generation of sufficient genomic information with cated the dynamic terpene biosynthetic activity of leaves deep coverage is required to gain insights into the regula- at this stage of development. PGT were purified from tory mechanism of terpene metabolism and glandular leaves of this stage and RNA isolated. The leaves of the trichome development. This will promote successful same stage were brushed to remove all trichomes and engineering for improved yields or to develop mint as a RNA extracted from them as controls (Additional file 1). platform for production of novel/altered terpenes. Mint is a well-suited plant for this as it is able to produce and Sequencing, de novo assembly and annotation of store large amount of oils within PGT instead of exuding transcriptome it on to the leaf surface. Storage within the PGTs also Three RNA libraries were prepared and sequenced by reduces the loss of volatile oils by emission into the Illumina technology. More than 100 million high quality atmosphere. reads of 101 base pairs (bp) were generated from PGT, High-throughput RNA sequencing (RNA-Seq) has in- leaf-PGT and leaf (Additional file 2). Using the Trinity creasingly become the technology of choice to generate a method [20] the sequence reads were finally assembled comprehensive and quantitative profile of the gene tran- into 40,587 non-redundant unigenes, spanning a total of scription pattern of a tissue. Here, we report comparative 101 Mb of sequence with a GC content of 43.14%. All analysis of RNA-seq transcriptome of different tissues of unigenes were longer than 200 bp. The N50 of the final spearmint-namely PGT, leaf minus PGT (leaf-PGT) and assembled transcripts was 1,774 bp. The unigenes were leaf. The transcriptome data provided a genome-wide annotated by performing BLASTX search against various insight into the metabolic ability of PGT. Comparison of protein databases. Among the 40,587 non-redundant PGT and leaf-PGT showed that 3,919 unigenes were dif- unigenes, 27,025 (67%) had at least one hit in BLASTX ferentially expressed in PGT (minimum 4 times more in search with E-value < = 1e-3. Functional classifications PGT when compared to leaf -PGT). Many of these were of Gene Ontology (GO) term of all unigenes were per- related to terpene production and other secondary metab- formed using Trinotate [20]. In order to calculate the ex- olite pathways. From the various terpene synthases (TPS) pression level for assembled transcripts, we first mapped transcripts identified, we functionally characterized 2 of reads onto them using bowtie [21]. RSEM (RNA-seq by these previously uncharacterized TPSs from mint and Expectation-Maximization) was used to estimate the found them to be sesquiterpene synthases. Key pathway abundance of assembled transcripts and to measure the unigene transcripts were verified by qRT-PCR. Our results expression level [22]. show the molecular specialisation of PGT for the production of different classes of metabolites. Overview of expression profile of spearmint PGT From the RNA seq data about 25,000 unigenes were ob- Results and discussion served to be expressed in spearmint PGT. The heat map Spearmint PGT and their development in Figure 3 exhibits some specific expression patterns to Spearmint leaves produce three different types of tri- PGT. Among this specific pattern for PGT we found chomes on their surfaces: non-glandular multicellular hair transcripts for terpene biosynthesis, lipid transfer pro- like, capitate glandular trichomes with a single secretory teins and interesting transcription factors like MYBs and head cell and PGTs whose secretory head is composed of WRKYs. Comparison of PGT and leaf-PGT showed that eight-cells with a single stalk and basal cell (Figure 1A). 3,919 unigenes were differentially expressed in PGT These PGT glands possess a large subcuticular storage (Additional file 3). These unigenes showed a minimum Jin et al. 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Figure 1 Trichomes on spearmint leaf. (A) Scanning electron microscope image of spearmint leaf showing three types of trichomes, a, Non glandular hairy trichome; b, Peltate glandular trichome (PGT); c, Capitate glandular trichome. (B) Process of secretion by PGT. a, presecretory stage; b, formation of storage cavity; c, secretion into the storage cavity; d, release of oil upon injury. The PGTs were stained with toulidine blue.

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Retention time (min) Figure 2 GC-MS of spearmint leaf showing the presence of monoterpenes and sesquiterpenes. Jin et al. BMC Plant Biology 2014, 14:292 Page 4 of 14 http://www.biomedcentral.com/1471-2229/14/292

they play a role in modification of cell wall layers to form the storage space remains to be investigated. To characterize biological processes specific for PGT, GO term was determined for all differentially expressed unigenes. Additionally, we identified unigenes whose expression was reduced in PGT by comparing leaf-PGT and PGT. These unigenes showed a minimum of 4 times reduction in expression level in PGT when compared to leaf-PGT. GO term was determined for them as well. Figure 4 shows the top 30 GO terms for the more abundant and less abundant unigenes. GO terms associated with ribosome biogenesis, ribosome structural genes and translation are highly represented in PGT, which could reflect the high protein biosynthetic activity of PGTs. Other terms included terpene metabolism and most of PGT Leaf-PGT Leaf the primary energy producing terms like glycolysis and tricarboxylic acid cycle. Furthermore, pentose phosphate Figure 3 Heat map of transcript expression in PGT, leaf-PGT and leaf. related term (oxidative) was also enriched in PGT. This term provides NADPH for biosynthetic processes such as fatty acid synthesis, cytochrome P450 mediated hydroxyl- of 4 times increase in expression level in PGT as com- ation and the assimilation of inorganic nitrogen [26]. pared to leaf-PGT. About 30% of these unigenes encoded These results indicate that the GO functions that provide either hypothetical proteins or remained unannotated. energy equivalents and redox cofactors are very active in Many of these unannotated unigenes showed none or PGT. Secretory trichomes are biosynthetically very active, minimal expression in leaf-PGT. They might represent and hence, there is a high energy requirement in these novel genes that are unique to PGTs development and di- cells. Unigenes from the GO terms of photosynthesis, vergent from other plants, whose genomes have been se- chlorophyll biosynthesis and starch biosynthesis were quenced. Data from proteomic analysis of spearmint PGT among the less abundant ones. This shows that our PGT also showed that the largest functional category of the sample preparation was pure and not contaminated with identified proteins was “unclear classification” and in- leaf tissues as mint PGTs are non-photosynthetic. cluded proteins with unknown functions [11]. The ab- Mint PGTs being non- photosynthetic and metabolically sence or low levels of some PGT-specific transcripts from very active would presumably rely on exogenous supply of leaf RNA seq data indicated the dilution of PGT-specific sucrose from underlying leaf tissues to use as carbon source RNAs among the total leaf RNAs and reaffirms the im- for energy production. We found several transcripts enco- portance of isolating these organs for analysis. ding enzymes for sucrose catabolism expressed more in Among the top 1000 differentially expressed unigenes, PGT like sucrose synthase and neutral/alkaline invertases we identified 16 TPSs, 18 cytochrome P450s, 5 lipid that are important for channelling carbon from sucrose in transfer proteins (LTPs), 20 transcription factors, 2 ATP- non-photosynthetic tissues [27]. These enzymes convert su- binding cassette (ABC) transporters and several tran- crosetohexosephosphates.PlastidsinthePGTsarethe scripts associated with cell wall. Cytochrome P450s are main sites of secondary metabolism. In contrast to chloro- involved in the hydroxylation of terpenes [23] and LTPs plasts, plastids of heterotrophic tissues have to rely on the have been suggested to be involved in intracellular trans- import of ATP and carbon to drive their metabolic pro- port and secretion of lipids and terpenes [9,24]. LTPs cesses. We checked our set of differentially expressed uni- were among the most abundant unigenes in PGT and genes to see if any known transporters are present. In most were confirmed by qRT-PCR (Additional file 4). The plants glucose 6-phosphate seems to be the preferred hex- abundance of these LTPs suggests their importance in ose phosphate taken up by nongreen plastids. The trans- PGTs metabolic function and development. ABC transporter proteins responsible for this import of carbon into porters are also proposed to be involved in the active trans- plastids are known as Glc6P–phosphate translocator (GPT) port of secondary metabolites [11,25]. The spearmint PGT and transcript similar to GPT was seen enriched in PGT is presumed to undergo cell wall modification to form sub- (about 30 times more in PGT). This carbon can be used for cuticular storage space [12]. Among the differentially starch biosynthesis or for the oxidative pentose phosphate expressed unigenes there were few that were related to cell pathway in plastids [28]. GO terms for oxidative pentose wall synthesis or modifications and a subset of these were phosphate pathways were seen enriched in PGT. Additio- confirmed by qRT-PCR (Additional files 4 and 5). Whether nally transcript similar to plastidic Phosphoenolpyruvate/ Jin et al. BMC Plant Biology 2014, 14:292 Page 5 of 14 http://www.biomedcentral.com/1471-2229/14/292

Figure 4 Top 30 GO annotation of more expressed unigenes (A) and less expressed unigenes (B). X-axis: log(1/P-value), P-value is the hypergeometic test result for each GO terms. phosphate translocator was found to be expressed more in different regulatory conditions [31]. Our analysis of tran- PGT. They are involved in the transport of phosphoenol- scriptome data of young leaves or PGT did not uncover pyruvate, an energy rich glycolytic intermediate from the any transcripts that matched those of the major known cytoplasm into the plastids [29]. Further, ATP generated trichome initiating gene transcripts from Arabidopsis, like either by glycolysis or by oxidative phosphorylation in mito- TRANSPARENT TESTA GLABRA1, GLABRA1, GLABRA3 chondria can be imported into nongreen plastids by plas- [32]. Either these genes are not expressed or are expressed tidic nucleotide transporter (NTT). Transcript similar to at a different developmental stage of leaves or PGT than NTT was also observed to be more abundant in PGT [29]. the stage used in this study. Table 1 shows the top 20 transcription factors that were significantly more abundant in Analysis of spearmint PGT transcription factors PGT when compared to leaf-PGT. Although the cloning and functional characterization of enzymes involved in terpene biosynthesis has been quite The MEP (2-Cmethyl-D-erythritol-4-phosphate) pathway is successful in various plants, knowledge about regulation more abundant in spearmint PGT than the MVA of these secretory trichome specific pathways is very (mevalonate) pathway rudimentary. Studies in peppermint show a close associ- The building blocks for all different classes of terpenes ation between enzyme activity and transcript abundance produced by plants are C5 units of isopentenyl diphos- for all gene/enzyme pairs, suggesting that, essential oil phate (IPP) and its allylic isomer dimethylallyl diphos- biosynthesis is primarily influenced at the transcriptional phate (DMAPP). They are generated either by plastidial level [12,17,18,30]. Hence, identification of transcription MEP or cytoplasmic MVA pathway. The MEP pathway re- factors that globally control metabolic pathway will pro- quires seven enzymes to synthesize IPP and DMAPP from vide an attractive strategy for engineering terpene pro- pyruvate and glyceraldehyde 3 phosphates which feed the duction. Similarly, knowledge about the development of monoterpene pathway [33]. From peppermint EST studies secretory glandular trichomes, the so-called factories it has been proposed that the active pathway for the for- of important terpenoid production, is very limited. Most mation of IPP/DMAPP in the PGT is the MEP pathway. of the transcription factors involved with trichome de- This is consistent with our analysis too where MEP path- velopment have been isolated from Arabidopsis, which way transcripts were more abundant in PGT than MVA. lacks secretory trichomes. Studies in tobacco and tomato High expression of MEP pathway transcripts correlates are beginning to show that multicellular secretory tri- well with the production of monoterpenes in PGT. It has chomes and unicellular trichomes of Arabidopsis are not been reported that 1-deoxy-D-xylulose-5-phosphate syn- homologous structures, and they likely develop under thase (DXS), the first enzyme of this pathway is important Jin et al. BMC Plant Biology 2014, 14:292 Page 6 of 14 http://www.biomedcentral.com/1471-2229/14/292

Table 1 Top 20 enriched TFs in PGT compared to leaf-PGT Name Leaf Leaf-PGT PGT FC Arabidopsis ID Description comp26629_c1 4.86 1.10 7.57 6.48 AT1G48000 myb comp29031_c0 7.08 4.51 10.93 6.42 AT2G26580 YABBY comp37102_c0 2.04 0.00 6.26 6.26 AT2G46150 Late embryogenesis abundant comp31772_c0 2.48 0.10 6.03 5.93 AT5G13080 WRKY comp31929_c0 3.97 1.52 7.27 5.75 AT2G47460 myb comp25071_c0 3.11 1.24 6.93 5.69 AT5G60910 MADS-box comp34871_c0 2.51 0.00 5.34 5.34 AT3G56850 Basic region/leucine zipper motif comp34759_c0 1.69 0.00 5.25 5.25 AT1G14600 Homeodomain-like comp43497_c1 2.42 0.53 5.36 4.84 AT1G68150 WRKY comp32776_c1 0.70 0.00 4.61 4.61 AT2G41690 Heat shock TF comp27387_c0 0.00 0.00 3.91 3.91 AT5G07680 NAC comp35105_c0 2.06 0.00 3.85 3.85 AT5G65790 myb comp25286_c0 2.13 0.00 3.80 3.80 AT5G01380 Homeodomain like TF comp30569_c1 2.32 1.08 4.85 3.77 AT5G66350 Lateral root primordium comp26625_c0 2.15 1.37 5.04 3.67 AT1G75390 bzip domain comp17332_c0 0.00 0.00 3.65 3.65 AT1G73230 btf3 comp32174_c0 3.56 2.22 5.70 3.48 AT5G13080 WRKY comp28852_c0 0.95 0.00 3.32 3.32 AT2G17770 Basic region/leucine zipper motif comp29336_c0 3.03 1.18 4.39 3.21 AT3G24860 Homeodmain-like comp42078_c1 3.48 0.77 3.84 3.08 AT4G32730 Homeodomain The value is log2 RESM value for each assembled TFs. FC is the log 2 fold change in PGT when compared to leaf-PGT. Arabidopsis ID is the homolog ID in Arabidopsis protein database. for the overall regulation of the pathway [34]. Multiple DXS In contrast to the MEP pathway, the transcript levels genes have been found in plants like Zea mays, Medicago of MVA enzymes were very low. For RNA seq expres- truncatula, Oryza sativa, Ginkgo biloba and Pinus sion levels of unigenes involved in this pathway refer densiflora and Picea abies [35-40]. In all these plants, two Additional file 6. Their expression was validated by qRT- or three candidate DXS genes have been reported. From PCR (Additional file 4). MVA pathway derived IPP is gen- ourdatawewereabletoidentify2different1-deoxy-D- erally believed to be used for the production of cytosolic xylulose-5-phosphate synthase (DXS) unigenes showing dif- sesquiterpenes, triterpenes and mitochondrial terpenes. In ferent levels of abundance in PGT. The number of genes addition to monoterpenes, mint produces a few sesquiter- coding for each MEP pathway enzyme varies from plant to penes although at much lower quantities than monoter- plant [33,41]. Presence of multiple genes with differential penes [9,42]. Lower level of MVA pathway can be one of tissue-specific expression levels might contribute towards the reasons. Interestingly, the transcripts of genes involved the regulation of the MEP pathway, in different organs of in the MEP pathway are also enriched in Artemisia annua the plant. Figure 5 shows the number of unigenes identified trichomes, glandular trichomes of Hops and in Snap- for each enzyme of the MEP pathway and their RNA seq dragon flowers where sesquiterpene metabolism domi- expression levels. In cases of enzymes with more than one nates [43-45] suggesting that the MEP pathway can also unigene, the unigene with the highest abundance in PGT feed sesquiterpene production. Studies with labelled sub- was taken into consideration. Their expression was further strate predict exchange of metabolites between the MVA validated by qRT-PCR (Additional file 4). From our RNA and MEP pathways [46,47]. How the IPP/DMAPP formed seq data and qRT-PCR analysis, DXR and MCT transcript by MEP pathway is utilized to synthesize sesquiterpenes in levels were low when compared to levels of other enzymes mint remains to be investigated. in MEP pathway. This might suggest that possibly these two enzymes are the rate limitingstepsofthispathway.A Monoterpene production is enriched in spearmint PGT possible option to explore in future will be to enhance the Subsequent condensation reactions between IPP and expression level of various rate limiting steps to enhance DMAPP are catalysed by GPP synthases (GPPS) that leads the production of terpenes. to the formation of geranyl diphosphate (GPP; C10) the Jin et al. BMC Plant Biology 2014, 14:292 Page 7 of 14 http://www.biomedcentral.com/1471-2229/14/292

themselves [48,49]. In spearmint too, we found unigenes for both the small and large subunits of GPP synthase that showed high expression in PGT. The major constituent of spearmint essential oil is (−) carvone which is synthesised from GPP in a three step reaction. Transcripts for all the three enzymes involved in above reaction, Limonene synthase (LS), Limonene-6-hydroxylase (L6OH) and carveol dehydrogenase (CD) were highly expressed in PGT and verified by q-RT-PCR (Figure 6 and Additional file 4). Interestingly, the precursor for menthol in peppermint and (−) carvone in spearmint is the same limonene. In peppermint it is oxygenated by (2)-4S-limonene-3-hydroxylase (L3OH) to form (2)-trans-isopiperitenol and it enters the menthol pathway whereas in spearmint limonene is oxygenated by (2)-4S-limonene- 6-hydroxylase (L6OH) to form (2)-trans-carveol. Both these enzymes show a 70% identity at the amino acid level with major dif- ferences localized to the presumptive active sites [50]. The spearmint L6OH transcript is highly expressed in PGT as expected but the full set of downstream redox enzymes isopiperitenone reductase, (+)-pulegone reductase, and menthone reductase involved in menthol pathway were also found but poorly expressed in PGT. A previous study has shown that (−) carvone is not an efficient substrate for the initial double-bond reductase, therefore (−) carvone accumulates in spearmint even though the downstream redox enzymes are present [11,51]. Hence, the abundance of a single enzyme L6OH instead of L3OH changes the final monoterpene produced. This shows how simple changes in the production of a single intermediate can result in drastic changes in the metabolic profiles. When compared to heterodimeric GPP synthase, transcripts for Figure 5 Expression level of unigenes involved in MEP farnesyl diphosphate synthase which is responsible for the pathway. The number in green represents the expression level of a particular unigene in PGT (log2 of estimate abundance of transcripts formation of farnesyl diphosphate precursor for sesquiter- by RSEM value). The number in red represents the fold change in penes, is expressed around 4 times less in PGT. Apart expression level when compared to leaf-PGT (log2 fold change from low MVA pathway, low levels of FPP synthase tran- between PGT and leaf-PGT). In cases of enzymes with more than scripts might also contribute to the reduced sesquiterpene one unigene, the unigene with the highest abundance was taken production in mint PGT. into consideration. The number in brackets represents the number of unigenes identified for each enzyme in the pathway. DXS: 1-deoxy- D-xylulose-5-phosphate (DXP) synthase; DXR: DXP reductoisomerase, Functional characterization of Terpene Synthases (TPS) MCT:MEP cytidyltransferase, CMK:4-(cytidine 5- diphospho)-2-C-methyl- from spearmint D-erythritol kinase MCS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate In plants, specific TPSs are responsible for the synthesis (ME-2,4cPP) synthase, HDS: 1-hydroxy-2-methyl-2-butenyl 4-diphosphate of various terpene molecules from the common precur- (HMBPP) synthase, HDR: HMBPP reductase, IPPI : Isopentenyl diphosphate (IPP,C5) Delta-isomerase. sors. Our transcriptome data provides a rich resource for identifying and functionally characterizing new TPSs from spearmint. From our enriched unigenes, we found precursor for monoterpenes. The conversion of IPP to 16 that were identified as terpene synthases; all of them DMAPP and its equilibrium is maintained by IPP isomer- were more than 1 kb and 10 of them were encoding full- ase (IPPI). In most plant species this enzyme is encoded by length open reading frames (ORFs). We found TPSs an- a single gene whereas Arabidopsis has two IPPI genes [33]. notated as limonene synthase, (E)-β-farnesene synthase, We found 2 IPPI unigenes in spearmint both enriched in bicyclogermacrene synthase and cis muuroladiene syn- PGT. Peppermint GPPS (Mp GPPS) is a two-component thase being preferentially expressed in PGT. However, heteromeric enzyme consisting of a large and a small sub- the exact functional annotation of a new TPS requires unit, and both the subunits are catalytically inactive by activity characterization of the recombinant protein. In Jin et al. BMC Plant Biology 2014, 14:292 Page 8 of 14 http://www.biomedcentral.com/1471-2229/14/292

Figure 6 Carvone biosynthesis pathway unigene levels. The number in green represents the expression level of a particular unigene in PGT (log2 of estimate abundance of transcripts by RSEM value). The number in red represents the fold change in expression level when compared to leaf-PGT (log2 fold change between PGT and leaf-PGT). In cases of enzymes with more than one unigene, the unigene with the highest abundance was taken into consideration. The number in brackets represents the number of unigenes identified for each enzyme in the pathway. LS: Limonene synthase, L6OH: Limonene-6-hydroxylase, CD: Carveol dehydrogenase. mint species, to our knowledge limonene synthase from MsTPS2 might be further metabolised endogenously by spearmint [52], (E)-β-farnesene synthase from peppermint the plant (data not shown). [42] and cis-muuroladiene synthase from black peppermint Sesquiterpene synthesis is thought to take place in the [53] have been previously characterised with respect to cytoplasm while monoterpene synthesis is believed to occur their functions. From our RNA seq data we chose to in plastids. Since our biochemical characterization shows characterize two unannotated full-length TPS. Phylogenetic that MsTPS1 and MsTPS2 are sesquiterpene synthases, we comparison (Additional file 7) showed that both MsTPS1 examined subcellular localization of these proteins in tran- and MsTPS2 belonged to the TPS-a subfamily of angio- sient studies in tobacco leaves. YFP-tagged MsTPSs were sperm sesquiterpene synthases. The main sesquiterpenes transiently expressed in N. benthamiana leaf cells by Agro- identified by GC-MS analysis in our spearmint variety were bacterium-mediated infiltration and visualized 3 dpi using (E)-β-farnesene, β-caryophyllene, α-caryophyllene (Humu- the YFP channel of a confocal microscope. Both the Ms lene), cis murola-3-5 diene, β-copaene, bicyclogermacrene TPSs showed cytoplasmic localization (Additional file 9) and bicyclosesquiphellandrene. and their sequence analysis also showed lack of plastid tar- To determine MsTPS1 and MsTPS2 enzymatic activities, geting sequences which further affirms that these TPSs are the full-length open reading frame encoding these enzymes sesquiterpene synthases. Therefore, either by sequence were overexpressed in E.coli, purified and used for in vitro similarity or by functional characterization we were able assays with GPP or FPP as substrate. In the presence of to identify all the major terpene synthases that are respon- FPP MsTPS1 catalysed the formation of β-caryophyllene sible for the formation of major spearmint essential oil in vitro (Figure 7A) whereas MsTPS2 produced a peak components. from FPP that was identified as one of β-cubebene/Germa- crene D/β-copaenebyGCMS(Figure7B).β-copaene was observed in our mint leaf GC-MS data suggesting that Spearmint PGTs as plants chemical defense organs MsTPS2 is most likely to be β-copaene synthase. Both Many of the secondary metabolites produced by glandular the TPSs failed to produce a peak with GPP as substrate trichomes play a role in plant defence. Majority of them (Figure 7A and B). Thus, our in vitro studies identified fall into the category of terpenes, phenylpropenes, flavo- them to be sesquiterpene synthases. Furthermore, transient noids, methyl ketones, acyl sugars and defensive proteins. Agrobacterium tumefaciens-mediated plant expression [54] Apart from having a rich terpene pathway, spearmint was used to investigate the terpenes produced by MsTPS1 PGT also shows presence of transcripts that are involved and MsTPS2 in planta.BothMsTPS1 and MSTPS2 under in the production of different secondary metabolites that the control of a 35S promoter were transiently expressed may have a role in plant defense. Transcripts encoding in N. benthamiana leaves by Agrobacterium-mediated infil- enzymes for phenylalanine ammonia lyase, cinnamate 4- tration. The compounds were analysed 3 dpi (days post- hydroxylase, and 4-coumarate CoA-ligase were seen infiltration) by GC-MS. Both TPSs failed to form any new expressed in PGT. These enzymes are involved in phenyl- peak when observed by GC-MS. Studies have shown that propanoid production. Presence of a variety of small mo- overexpressing enzyme 3-Hydroxy-3-Methylglutaryl Coen- lecular weight phenylpropanoids like caffeic, rosmarinic zyme A Reductase (HMGR), a rate limiting step of the and ferulic acids has been detected in leaves of different mevalonate pathway increases heterologous plant sesqui- mint germplasm [56]. Transcripts similar to Caffeate O- terpene production [55]. Accordingly, both the TPSs were methyltransferase, an enzyme required for the conversion coexpressed with HMGR in planta to observe the produc- of caffeic acid into ferulic acid was preferentially expressed tion of sesquiterpenes. MsTPS1 with HMGR produced β- in PGT. Chalcone flavanone isomerase an enzyme in the caryophyllene as the major peak and α-caryophyllene and flavonoid pathway in plants was also preferentially caryophyllene oxide as minor peaks Additional file 8. expressed in PGT. Unigenes encoding transcripts similar MsTPS2 even with HMGR failed to produce any new to plant invertase/pectin methylesterase inhibitors were peaks in planta suggesting that the compound formed by highly expressed in PGT which are important to defend Jin et al. BMC Plant Biology 2014, 14:292 Page 9 of 14 http://www.biomedcentral.com/1471-2229/14/292

(x100,000) (x100,000) A + FPP 9 9 GST-MsTPS1 GST + FPP + GPP 8 8 GST + GPP 7 7 6 6 5

5 abundance

4 4

3 3

Relative 2 Relative abundance 2

1 1

0 0 17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8 17.9 18.0 18.1 18.2 18.3 17.1 17.2 17.3 17.4 17.5 17.6 17.7 17.8 17.9 18.0 18.1 18.2 18.3 Retention time (min) Retention time (min)

Peak Ion Abundance

β-Caryophyllene, 32.9% Ion Abundance

m/z

(x100,000) B 1.8 (x100,000) 1.8 GST + FPP + FPP 1.6 GST-MsTPS2 1.6 + GPP GST + GPP 1.4 1.4 1.2 1.2 1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4 Relative abundance Relative abundance

0.2 0.2

0 0 18.0 18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8 18.9 19.0 19.1 19.2 18.0 18.1 18.2 18.3 18.4 18.5 18.6 18.7 18.8 18.9 19.0 19.1 19.2 Retention time (min) Retention time (min)

Peak Germacrene D, 19.4% Ion Abundance Ion Abundance β-Cubebene, 23% β-copaene, 16.4% Ion Abundance Ion Abundance

m/z m/z Figure 7 (See legend on next page.) Jin et al. BMC Plant Biology 2014, 14:292 Page 10 of 14 http://www.biomedcentral.com/1471-2229/14/292

(See figure on previous page.) Figure 7 In vitro enzymatic assays of recombinant MsTPSs. GST-tagged MsTPS recombinant enzymes were purified by glutathione-based affinity chromatography and used for in vitro assays with GPP or FPP as substrate. The final products were analysed by GC-MS. The peaks marked with an arrow in the GC traces were compared with the reference of the mass spectra library. Mass spectra for the peaks formed with FPP are shown at bottom of figure. m/z, mass-to-charge ratio, (A) Left panel, β-caryophyllene formation by GST-MsTPS1 with FPP; Right panel, the control GC-MS analyses of GST with GPP or FPP. (B) Left panel, β-cubebene/Germacrene D or β-copaene formation by GST-MsTPS2 with FPP; Right panel, the control GC-MS analyses of GST with GPP or FPP. against plant pathogens [57,58]. All the above transcripts Methods were verified by qRT-PCR (Additional file 4). Plant material, PGT and RNA isolation Oxylipins are large family of biologically active oxidized Commercial spearmint variety was grown in green house fatty acid-derivatives that are important for plant defense under natural light conditions. 1–2 cm leaves were col- responses. They are produced mainly by the combined lected in ice cold imbibition buffer similar to described action of lipases, lipoxygenases (LOXs) and members of in [9] in a 50 ml falcon tube. After 1 hour of soaking, cytochrome P450 (CYP74) family specialized in metaboli- the leaves (approximately 3 g) were transferred to a fresh zing fatty acids hydroperoxides (HPs). HPs form the initial falcon tube containing the extraction buffer as described substrates for different branches of the oxylipin pathway in [9]. Trichomes were isolated by glass bead abrasion [59-62]. We found enzymes involved in oxidation of fatty method. Glass beads (Sigma 425–600 μm) were added acids expressed more in PGTs. Transcripts similar to to the falcon tube and vortexed for 30 sec twice with a phospholipase A, lipoxygenase and allene oxide synthase 1 min resting period on ice. Then the mixture was care- and allene oxide cyclase were enriched in PGT. Allene fully passed through cell strainer (100 μM) to remove oxide synthase and allene oxide cyclase are members of the cell wall debris and hairy trichomes and the flow the cytochrome P450 (CYP74) family and are involved in through collected. This was again sieved using 40 μM the LOX pathways [63,64]. All the above mentioned tran- cell strainer to allow passage of capitate glandular trich- scripts showed a minimum of 8 times enrichment in PGT ome and collection of PGT above. The accumulated and were confirmed by qRT-PCR. However to understand PGT on top of 40 μM strainer was washed in isolation the role of these enzymes in plant defence and exact nature buffer few times and finally collected using RNAase free of oxylipins formed by them, further characterization is es- water into a 1.5 ml centrifuge tube. The collected PGT sential, especially in terms of their positional specificity and was then spun and excess water removed and immediately substrate specificity. Plants possess both basal and indu- frozen in liquid nitrogen for further use. We randomly cible mechanisms to defend themselves against pathogens. counted around 500 isolated PGT to get an estimate on The transcriptome data reflects the basal or constitutive sample purity and found 26 hair-like trichomes, mostly state of defense mechanism existing in spearmint PGT. broken, and 4 CPT indicating a good purity level of iso- The changes in transcript abundance in all the above men- lated PGT. Same stage leaves were brushed in imbibition tioned genes on stress induction will provide a better un- buffer to remove trichomes and checked under dissection derstanding of how PGT act as chemical defence organs. microscope. Total RNA was extracted from PGT, leaf and leaf-PGT using the Spectrum™ Plant total RNA kit from Conclusions Sigma. The quality of RNA was checked by measuring the Availability of extensive genome resources in non-model ratio of OD260 to OD280 and the integrity was assessed by plant Mentha is scarce. This is the first attempt at the de measuring the RNA Integrity Number (RIN) number novo sequencing and assembly of transcriptomes derived using Agilent 2100 bioanalyser. from various tissues of Mentha spicata using NGS. Com- parison of PGT and leaf-PGT led to the identification of Sequencing and assembly 3,919 differentially expressed unigenes. Analysis of these The RNA libraries were prepared using the TruSeq RNA unigenes provides an insight into the gene expression pat- Sample Preparation Kits v2, set A (RS-122-2001, Illumina tern and biological processes active in spearmint PGT. Inc.) according to manufacturer’s instructions. The quality Further identification of various unknown PGT specific and size of cDNA libraries for sequencing were checked unigenes should facilitate new gene discovery. Our tran- using the Agilent 2200 TapeStation system (Agilent Inc.). scriptome data provides essential information for future The libraries were run on single lanes for 100 cycles ™ genetic studies in spearmint. It will also help in developing (paired-end) on Hiseq 2000 (illumine Inc.), individually. strategies to engineer the terpene metabolic process that Raw reads were analysed by FastQC [65] for their quality can be further extrapolated to other commercially impor- and found to high quality reads with Q > 20. The Trinity tant plants similar to mint where no genomic resources method [20] was used for de novo assembly of the raw are available. reads to generate unigenes. Functions of the unigenes Jin et al. BMC Plant Biology 2014, 14:292 Page 11 of 14 http://www.biomedcentral.com/1471-2229/14/292

were annotated based on sequence similarities to se- was digested by XbaI and AscI, and cloned into quences in the public nr database (National Centre for pCAMBIA1300-3HA. In vivo characterization was done Biotechnology Information) and also the protein sequence in Nicotianana benthamiana leaves by Agrobacterium- databases from Arabidopsis thaliana, Vitis vinifera and mediated infiltration. Initially Agrobacterium strain har- Oryza sativa. The GO terms were retrieved by Trinote bouring MsTPS1 was used alone and then strain carrying from the Gene Ontology database [20]. AtHMGR1 was mixed with MSTPS1 strain and coinfil- trated. Overnight cultures of Agrobacterium grown at Preparation of recombinant proteins and in vitro enzyme 28°C were harvested. It was resuspended to a final con- assay centration to an absorbance of 1.0 at 600 nm in a solu- The full-length cDNAs of MsTPS1 and MsTPS2 were tion containing 10 mM MgCl2, 10 mM MES pH 5.6 and amplified with the following primer sets from PGT de- 100 μM acetosyringone. After 2 hour incubation at room rived cDNA. MsTPS1:5′-CACCATGGAAATTCCTGC temperature, the Agrobacterium mixture was injected ACCGGTTTCGGCTTA-3′ and 5′-AACTGTTAGGGG into N. benthamiana leaves using a needleless syringe. ATCAACGAGTATGGATTTGATC-3′; and for MsTPS2: Infiltrated plants were incubated in the growth chamber and 5′-5′CACCATGGCTGAAATCTGTGCGTCGGC at 24°C for 3 days or 6 days. Three to four infiltrated TGCT-3′ and 5′ GTGCAGGGGATCTACGAGCACGG leaves were taken for GC-MS analysis. ATTGAAT-3′. To construct the vectors for the production of recombinant GST-tagged proteins, the PCR- GC-MS analysis method amplified MsTPS1 and MsTPS2 cDNAs were inserted into For extraction about 4–6 leaves were ground to a fine pGEX-4 T-1 (GE Healthcare Life Sciences) to generate powder using liquid nitrogen and the powder homoge- GST-MsTPS1 and GST-MsTPS2, respectively. Both these nised in 500 μl ethyl acetate including 1 μl (10 mg/ml) constructs were transformed into E.coli BL21-CodonPlus of camphor as internal standard and incubated at least (DE3)-RIPL (Stratagene), and treated with 0.2 mM isopro- 2 h at room temperature with shaking. This mixture was pyl 1-thio-β-D-galactopyranoside (IPTG) at 20°C for centrifuged for 5 min at 12,000 rpm. The top organic layer overnight to induce GST-tagged protein expression. was transferred to a new tube and dehydrated using an- The harvested cell pellets were resuspended in lysis hydrous Na2SO4. The samples were analysed using GCMS buffer(20mMTris,pH7.4,150mMNaCl,10mMβ- (Agilent Technologies 7890A with 5975C inert Mass-spe- mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, ctro Detector with tripe axis detector). 2 μl of samples and protease inhibitors cocktail) and broken by sonic- were injected and separation was achieved with a tem- ation. The clarified lysate was collected by centrifuga- perature program of 50°C for 1 min and increased at a tion and incubated with glutathione Sepharose 4B resin rate of 8°C/min to 300°C and held for 5 min, on a 30 m (GE Healthcare Life Sciences) at 4°C overnight. Pro- HP-5 MS column (Agilent Technologies). teins bound to glutathione Sepharose 4B resin were washed with the purification buffer, eluted from the Subcellular localization of TPSs- column with 10 mM glutathione and dialyzed against a The full-length cDNAs of MsTPS1 and MsTPS2 were buffer containing 25 mM HEPES, pH 7.5, 100 mM KCl, amplified with the following primer sets from PGT de- 1 mM DTT, and 5% glycerol. rived cDNA. MsTPS1:5′-CACCATGGAAATTCCTGC For in vitro enzyme assay for terpene synthase activity, ACCGGTTTCGGCTTA-3′ and 5′-AACTGTTAGGGG 10 μg of recombinant protein was used in final volume ATCAACGAGTATGGATTTGATC-3′; and for MsTPS2: of 500 μl of reaction buffer (25 mM HEPES, pH 7.5, and 5′-5′CACCATGGCTGAAATCTGTGCGTCGGCT 100 mM KCl, 7.5 mM MgCl2, 5 mM DTT, and 5% gly- GCT-3′ and 5′ GTGCAGGGGATCTACGAGCACGGA cerol) with 10 μg of substrate (farnesyl diphosphate, FPP TTGAAT-3′. Both the cDNAs were cloned into pEN or geranyl diphosphate, GPP; Sigma). Overlaid 250 μlof TR/D-TOPO vector (Invitrogen), and then transferred hexane to trap volatile products and incubated at 30°C into pBA-DC-YFP [66] which contains the CaMV for 2 h. Extracts were analysed by GC-MS (Agilent). 35S promoter and C-terminal in frame YFP, to generate MsTPS1-YFP and MsTPS2-YFP, respectively. The MsTPS1- In vivo characterization of MsTPS1 YFP or Ms-TPS2-YFP constructs were introduced into A. The full length cDNA encoding Arabidopsis 3-hydroxy- tumefaciens strain GV3101 by electroporation. YFP-tagged 3-methylglutaryl coenzyme A reductase (AtHMGR1, MsTPSs were co-expressed with CFP or alone to confirm At1g76490) was amplified using two specific primers, the cellular localization of MSTPS-YFP. CFP expression AtHMGR-F-XbaI (5′-AACTCTAGAATGAAGAAAAA was used as a cytoplasmic maker protein. Overnight cul- GCAAGCTGGTCCCCAACAGA-3′) and AtHMGR-R- tures of Agrobacterium grown at 28°C were harvested. It AscI primers (5′-AAAGGCGCGCCTGTTGTTGTTGT was resuspended to a final concentration to an absorbance TGTCGTTGTCGTT-3′). The PCR-amplified product of 1.0 at 600 nm in a solution containing 10 mM MgCl2, Jin et al. BMC Plant Biology 2014, 14:292 Page 12 of 14 http://www.biomedcentral.com/1471-2229/14/292

μ 10 mM MES pH 5.6 and 100 M acetosyringone. After Additional file 4: qRT-PCR analyses of described unigenes. DXS: 2 hour incubation at room temperature, the Agrobacterium 1-deoxy-D-xylulose-5-phosphate (DXP) synthase; DXR: DXP reductoisomerase, mixture was injected into N. benthamiana leaves using a MCT:MEP cytidyltransferase, CMK:4-(cytidine 5- diphospho)-2-C-methyl-D- erythritol kinase MCS: 2-C-methyl-D-erythritol 2,4-cyclodiphosphate needleless syringe. Infiltrated tobacco plants were placed in (ME-2,4cPP) synthase, HDS: 1-hydroxy-2-methyl-2-butenyl 4-diphosphate the growth chamber at 24°C for 3 days. Fluorescence sig- (HMBPP) synthase, HDR: HMBPP reductase, IPPI : Isopentenyl diphosphate nals were detected by a confocal scanning laser microscopy (IPP,C5) Delta-isomerase, GPPS: geranyl diphosphate synthase, LS: limonene synthase, L6OH: Limonene-6-hydroxylase, CD: carveol dehydrogenase, LTP: (Carl Zeiss LSM 5 Exciter) with a standard filter set. Lipid transfer protein. Additional file 5: Subset of cell wall related unigenes more Quantitative real time PCR (qRT-PCR) abundant in PGT. The qRT-PCR was employed to validate gene expression Additional file 6: Expression levels of transcripts involved in MVA μ pathway. The number in green represents the expression level of a pattern of transcriptome analysis. Approximately 1 g particular unigene in PGT (log2 of estimate abundance of transcripts by RNA was used for cDNA synthesis with iScript super- RSEM value). The number in red represents the fold change in expression mix (Bio-rad). The reaction was carried out by incuba- level when compared to leaf-PGT (log2 fold change between PGT and leaf-PGT). In cases of enzymes with more than one unigene, the unigene tion for priming at 25°C for 5 min, followed by reverse with the highest abundance was taken into consideration. The number in transcription at 42°C for 30 min and inactivation at 85°C brackets represents the number of unigenes identified for each enzyme for 5 min. cDNA was stored at −20°C till further used. in the pathway. The qRT-PCR reactions were performed in 384-well Additional file 7: Phylogenetic analysis of full-length MsTPS1 and MsTPS2 to other plant terpene synthases. The neighbour-joining tree PCR plate using ABI PRISM 900HT real-time PCR sys- was created using MEGA5.2 program from an alignment of Mentha piperita tem and KAPA SYBR fast master mix (KAPA Biosys- (E)-β-farnesene synthase (AF024615), Lycopersicon esculentum germacrene C tems).PCR reactions were performed using 0.3 μl of the synthase (AF035630), Salvia officinalis 1,8-cineole synthase (AF051899), cDNA in a total of 5 μl reaction volume and cycling pro- Mentha spicata 4S-limonene synthase (L13459), Perilla citriodora limonene synthase (AF241790), Salvia officinalis (+)-bornyl diphosphate synthase file was 95°C for 10 min, 40 cycles of 95°C for 15 s and (AF051900), Salvia officinalis (+)-sabinene synthase (AF051901), Perilla 60°C for 60 s. After thermal cycles, the dissociation ana- frutescens linalool synthase (AF444798), Citrus limon (+)-limonene synthase 1 lysis (melting curve) was carried out to confirm specific (AF514287), Arabidopsis thaliana myrcene/ocimene synthase (AF178535), amplification of PCR reaction. All reactions were per- Artemisia annua (3R)-linalool synthase (AF154124), Antirrhinum majus nerolidol/ linalool synthase 1 (EF433761), Antirrhinum majus (E)- β -ocimene synthase formed in triplicate with three biological replicates, (AY195607), Solanum lycopersicum copalyl diphosphate synthase (AB015675), including non-template control. The threshold cycle Cucurbita maxima copalyl diphosphate synthase (AF049905), Zea mays terpenesynthase1(AF529266),Cucurbita maxima copalyl diphosphate (CT) value of gene is the cycle number required for synthase 1 (AF049905), Abies grandis δ-selinene synthase (U92266), Abies SYBR Green fluorescence signal to reach the threshold grandis (−)-4S-limonene synthase (AF006193), Abies grandis pinene synthase level during the exponential phase for detecting the (U87909), Abies grandis terpinolene synthase (AF139206), Abies grandis amount of accumulated nucleic acid [67]. In current myrcene synthase (U87908), Picea abies E-α-bisabolene synthase study, elongation factor 1 (ef1) was used as internal con- (AY473619), Cichorium intybus germacrene A synthase short form (AF498000), Lactuca sativa germacrene A synthase LTC1 (AF489964), trol, due to its stable expression in plant [68] and it also Solidago canadensis germacrene D synthase (AJ583447), Solidago showed similar expression levels in all the tissues in our canadensis germacrene A synthase (AJ304452), Artemisia annua β RNA seq data. Comparative delta CT values of target -caryophyllene synthase QHS1 (AF472361), Artemisia annua amorpha-4, β genes to ef1 were taken as relative expression among dif- 11-diene synthase (JQ319661), Artemisia annua (E)- -farnesene synthase (AY835398), Arabidopsis thaliana copalyl diphosphate synthase ferent tissues. The amount of target gene, normalized to (NM_116512), Arabidopsis thaliana ent-kaurene synthase GA2 (AF034774), -(C target gene-C ef1) ef1 gene, was calculated by 2 T T . Results Stevia rebaudiana copalyl pyrophosphate synthase (AF034545), Stevia were represented as mean ± SD. The unigene names and rebaudiana kaurene synthase (AF097310). The scale bar indicates the sequence of primers used are listed in Additional file 10. number of amino acid substitutions per site. Five TPS subfamilies, a to e are based on the taxonomic distribution of the TPS families [69]. Additional file 8: GC-MS data generated from in vivo characterization Availability of supporting data of MsTPS1. MsTPS1 with or without HMGR was transiently expressed in N. benthamiana leaves by Agrobacterium-mediated infiltration. The compounds The raw RNA seq data supporting the result of this art- were analysed 3 dpi (days post-infiltration) by GC-MS. Numbered peaks were icle is available in the DDBJ: DNA Data Bank of Japan, identified by the reference of the mass spectra library and the mass spectra with accession number is: DRA001856 (http://trace.ddbj. of compounds are shown at right side. nig.ac.jp/DRASearch/submission?acc=DRA001856). Additional file 9: Subcellular localization of MsTPS1 and MsTPS2 in N. benthamiana leaf. (A) YFP-tagged MsTPSs were transiently expressed in N. benthamiana leaf cells by Agrobacterium-mediated infiltration and Additional files visualized 3 dpi using YFP channel of a confocal microscope. (B) YFP- tagged MsTPSs were co-expressed with CFP to confirm the cytoplasmic localization of MSTPS-YFP. CFP expression is used as a cytoplasmic Additional file 1: Tissues from which RNA were isolated. a, isolated maker protein. CFP: CFP channel image, YFP: YFP channel image, PGT; b, leaf-PGT; c, leaf. Auto: chlorophyll auto fluorescence, Light: light microscope image, Additional file 2: Quality of reads and statistics of sequencing. Merged: merged image between Light and YFP. Additional file 3: Differentially expressed genes in PGT. Additional file 10: Primer sequences used for qRT-PCR analysis. Jin et al. BMC Plant Biology 2014, 14:292 Page 13 of 14 http://www.biomedcentral.com/1471-2229/14/292

Abbreviations 7. Saharkhiz MJ, Motamedi M, Zomorodian K, Pakshir K, Miri R, Hemyari K: PGT: Peltate glandular trichomes; TPS: Terpene synthases; EST: Expressed Chemical composition, antifungal and antibiofilm activities of the sequence tags; GC-MS: Gas chromatography–mass spectrometry; RSEM: RNA- essential oil of mentha piperita L. ISRN Phar 2012, 2012:718645. seq by Expectation-Maximization; LTP: Lipid transfer proteins; GPT: Glc6P– 8. Chauhan RS, Kaul MK, Shahi AK, Kumar A, Ram G, Tawa A: Chemical phosphate translocator; NTT: Nucleotide transporter; MVA: Mevalonate pathway; composition of essential oils in Mentha spicata L. accession [IIIM(J)26] MEP: 2-Cmethyl-D-erythritol-4-phosphate; IPP: Isopentenyl diphosphate; from North-West Himalayan region, India. Ind Crops Prod 2009, DMAPP: Dimethylallyl diphosphate; GPP: Geranyl diphosphate; FPP: Farnesyl 29(2–3):654–656. diphosphate; GGPP: Geranylgeranyl diphosphate; DXS: 1-deoxy-D-xylulose-5- 9. Lange BM, Wildung MR, Stauber EJ, Sanchez C, Pouchnik D, Croteau R: phosphate synthase; DXR: 1-deoxy-D-xylulose 5-phosphate reductoisomerase; Probing essential oil biosynthesis and secretion by functional evaluation MCT: 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK: 4-(cytidine of expressed sequence tags from mint glandular trichomes. Proc Natl 5-diphospho)-2-C-methyl-D-erythritol kinase; MCS: 2-C-methyl-D-erythritol Acad Sci U S A 2000, 97(6):2934–2939. 2,4-cyclodiphosphate synthase; HDS: 1-hydroxy-2-methyl-2-butenyl 10. Croteau RB, Davis EM, Ringer KL, Wildung MR: (−)-Menthol biosynthesis 4-diphosphate synthase; HDR: 1-hydroxy-2-methyl-2-butenyl 4-diphosphate and molecular genetics. Naturwissenschaften 2005, 92(12):562–577. reductase; IPPI: Isopentenyl diphosphate isomerase; GPPS: Geranyl 11. Champagne A, Boutry M: Proteomic snapshot of spearmint (Mentha diphosphate synthase; LS: Limonene synthase; L6OH: Limonene-6- spicata L.) leaf trichomes: a genuine terpenoid factory. Proteomics 2013, hydroxylase; CD: Carveol dehydrogenase; ORF: Open reading frame; 13(22):3327–3332. dpi: Days post-infiltration; HMGR: Hydroxy-3-Methylglutaryl Coenzyme A 12. Turner GW, Gershenzon J, Croteau RB: Development of peltate glandular Reductase; LOX: Lipoxygenases. trichomes of peppermint. Plant Physiol 2000, 124(2):665–680. 13. Burbott AJ, Loomis WD: Evidence for metabolic turnover of monoterpenes in peppermint. Plant Physiol 1969, 44(2):173–179. Competing interests 14. Croteau R, Martinkus C: Metabolism of monoterpenes: demonstration The authors declare that they have no competing interests. of (+)-neomenthyl-beta-d-glucoside as a major metabolite of (−)-menthone in peppermint (mentha piperita). Plant Physiol 1979, 64(2):169–175. Authors’ contributions 15. Maffei M, Gallino M, Sacco T: Glandular trichomes and essential oils of JJ, JL and LSW performed the bioinformatics analysis of the RNA seq data, developing leaves in mentha viridis lavanduliodora. Planta Med 1986, DNP did the trichome isolation and RNA preparation of spearmint tissues, 52(03):187–193. QW performed the qRT-PCR analysis, MJK and ICJ conducted the TPS 16. Brun N, Colson M, Perrin A, Voirin B: Chemical and morphological studies characterization; JLY did the GC-MS studies. NHC and RS developed the of the effects of ageing on monoterpene composition in Mentha × project and wrote the manuscript. All authors read and approved the final piperita leaves. Can J Bot 1991, 69(10):2271–2278. manuscript. 17. Gershenzon J, McConkey ME, Croteau RB: Regulation of monoterpene accumulation in leaves of peppermint. Plant Physiol 2000, 122(1):205–214. Acknowledgements 18. McConkey ME, Gershenzon J, Croteau RB: Developmental regulation of We thank Hufeng Zhou for helping with bioinformatics analysis. We thank monoterpene biosynthesis in the glandular trichomes of peppermint. the Rockefeller University sequencing facility and Dr. Hui-Wen Wu for RNA Plant Physiol 2000, 122(1):215–224. seq. Prasanna Nori Venkatesh for maintaining the greenhouse. This research 19. Kjonaas R, Croteau R: Demonstration that limonene is the first cyclic was funded by a grant from Singapore National Research Foundation intermediate in the biosynthesis of oxygenated p-menthane monoterpenes (Competitive Research Programme Award No: NRF-CRP8-2011-02) to SR, ICJ in Mentha piperita and other Mentha species. Arch Biochem Biophys 1983, and NHC and a grant from Singapore Millennium Foundation to NHC. JJ was 220(1):79–89. partially supported by a Singapore Ministry of Education Tier-2 grant, 20. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis MOE2009-T2-2-004. 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