Presence of monoterpene synthase in four Labiatae species and Solid-Phase Microextraction- Gas chromatography-Mass Spectroscopy analysis of their aroma profiles.

BACKGROUND: The family Lamiaceae (Labiatae) has included some medicinal plants. some monoterpene synthases, including linalool and limonene synthases, have been cloned and functionally characterized from several plants of Labiatae family.
MATERIALS AND METHODS: In this study, presence of linalool and limonene synthases, in four species of Labiatae family including Nepeta cataria, Lavandula angustifolia, Hyssopus officinalis and Salvia sclarea has been determined by molecular biological techniques together with the Head space SPME - GC-MS analysis of the aroma profile of these species.
RESULTS: Indicated that none of the plant species produced distinguishable bands with primer pairs related to d-limonene synthase. Distinguishable bands around 1800 bp in cDNA samples of L. angustifolia, H. officinalis and S. sclarea were observed regarding to the presence of linalool synthase. Head space SPME-GC-MS analysis of the aroma profiles of the above-mentioned plants showed that linalool (31.0%), linalyl acetate (18.2%), were found as the major compounds of L. angustifolia, while geraniol (5.5%), nerol (34.0%) and α- citral (52.0%) were identified as the main compounds of the N. cataria. The major components of H. officinalis and S. sclarea oils were determined as cis-pinocamphone (57.3%), and linalool (19.0%), linalyl acetate (51.5%), respectively.
CONCLUSION: H. officinalis was rich of cyclic monoterpenes, L. angustifolia, N. cataria and S. sclarea showed considerable amount of linear monoterpenes. The aroma profile of the above-mentioned plants contained low concentration of sesquiterpenes except N. cataria, which indicated no sesquiterpene. The profiles of the main components of these plants are in agreement with molecular assays.

INTRODUCTION

The family Labiateae (Lamiaceae) has 180 genera and about 3500 species. The aerial parts of these plants contain flavonoids, triterpenoids, and monoterpenes, particularly in the flowers and leaves.[123] Essential oils of these plants consist of various monoterpenes and sesquiterpenes, as well as their derivatives, such as alcohols, aldehydes, esters, and acetates.[456] Terpenoids are formed from the condensation of dimethylallyl pyrophosphate (DMAPP) and isopentyl pyrophosphate (IPP), to give geranyl diphosphate (GPP), farnesyl diphosphate (FPP), or geranyl geranyl diphosphate (GGPP).[789] Terpene synthases in plant kingdom may fall into two categories. First class (e.g., limonene synthase) involves in ionization of allylic diphosphate to initiate the reaction, but second class (e.g., copalyl diphosphate synthase (CPS)) acts in double-bond protonation of substrate.[10]

Among the terpene synthases, cyclases enzymes, such as limonene synthase (LMS), have been focused and studied in diverse plant species. LMS contains approximately 600 amino acids and is responsible for catalyzing the formation of limonene.[10] Limonene is a simple cyclic monoterpene, with a chiral center and two enantiomers (d and l). Although various d- and l-limonene synthase genes have been identified, there are a few reports about the relationship between gene transcription and biosynthesis during plant growth.[11] Linalool synthase (LIS), involves in biosynthesis of linalool as an acyclic monoterpene alcohol, is not simply classified on the basis of its amino acid sequence, because N-terminal part reveals higher sequence similarity to CPS but C-terminal exhibits higher similarity to LMS.[1213] Intron positions in LMS-type terpene cyclase genes are very similar to each other.[14]

So far, some monoterpene synthases, including linalool and limonene synthases, have been cloned and functionally characterized from several plants of Labiatae family.[1516] In the present study, we aimed to determine the presence of two monoterpene synthases, linalool and limonene synthases, in four species of Labiatae family including Nepeta cataria, Lavandula angustifolia, Hyssopus officinalis and Salvia sclarea together with the Head space Solid-phase Microextraction - Gas chromatography - Mass spectroscopy analysis of the aroma profile of these species.

MATERIALS AND METHODS

Experimental

Chemical reagents and solvents were purchased from Merck Co. (Germany). Agarose and 1kb DNA size marker were prepared from Invitrogen Co. (UK). RNeasy Plant Mini Kit was prepared from Qiagen (USA). Polymerase chain reactions were performed on a Primus 25 (Peqlab, Germany) thermal cycler. Primers were produced in Cinnagene (Iran).

Plant material

All the plants mentioned here were grown in the Herboratum of Faculty of Pharmacy, Tehran University of Medical Sciences and identified by Dr. Gholamreza Amin (Department of Pharmacognosy) as Nepeta cataria, Lavandula angustifolia, Hyssopus officinalis and Salvia sclarea. Young leaves of each species were harvested (during June, 2010) from a plant grown outside in a mini-garden under natural conditions.

Primer designation

Protein sequences of linalool and limonene synthases from Mentha spicata (Gen-Bank Accession No. AAC37366), Mentha citrate (AAL99381), Perilla frutescens (AAL38029), A. thaliana (AAO85533) and Lavandula angustifolia (ABB73045, ABB73044) were aligned with free ClustalW software and revealed several conserved regions. Based on the DNA and peptide sequences the different primers have been designed and synthesized [Table 1].

Table 1

Primers designed according to different terpene synthases gene sequences

cDNA Preparation and PCR

About 200 mg of each plant's leaves were frozen in liquid nitrogen and ground into a fine powder. Total RNA was extracted using “RNeasy Plant Mini Kit” and reverse transcribed with oligo (dT) primer [ad: 5’-GCT GTC AAC GAT ACG CTA CGT AAC GGC ATG ACA GTG TTT TTT TTT TTT TTT TTT-3’] designed to have an adaptor sequence at the 5’-end to obtain the cDNA. The obtained cDNAs were employed as the templates in various PCRs with Taq and/or KOD Dash DNA polymerases. The temperature program was started at 94°C (3 min), followed by 40 cycles: 94°C for 30 s, 46°C for 30 s (different annealing temperatures were used for each pair of primers) and 72°C for 1 min, then 72°C for 2 min. Elongating times were different (30-60 s) based on the expected length of the amplified fragment. The PCR was performed by DNA polymerase (0.2 μL), each degenerate forward and reverse primers (0.3 μL, 20 pmol), dNTP (0.1mM, 2 μL), DNA template (1 μL) and appropriate amounts of recommended buffer, DMSO and water. The partial size of monoterpene synthase sequences was estimated by gel electrophoresis. PCR products were run on a 1% (w/v) agarose gel along with a 1 kb DNA size marker, stained by ethidium bromide (0.5 μg/ml) and visualized in a gel documentation system.

SPME- GC-MS analysis

Head space Solid-phase Microextraction (SPME) coupled to gas chromatography and mass spectrometry has been applied for analyzing the essential oil directly evaporated from young leaves of four species. GC-MS was performed on a cross-linked 5% methyl phenyl siloxane (HP-5, 30 m × 0.25-mm i.d., 0.25-μm film thickness), carrier gas, He; split ratio, 1:15; quadruple mass spectrometer Hewlett-Packard 6890) operating at 70 eV ionization energy. In order to obtain the retention index for each compound, normal alkanes (C8-C25) were injected at the same temperature and condition. The components were identified by comparison of their retention indices (RI, DB-5) and mass fragmentation with those reported in the literature.[20] Percentage of each component was calculated on the basis of the peak area.

RESULTS AND DISCUSSION

In this investigation, PCR method was employed with different primers designed regarding to the conserved amino acid sequence in various Labiatae plant terpene synthases, in order to reveal the presence of limonene and linalool synthases in four Labiatae species: N. cataria, L. angustifolia, H. officinalis and S. sclarea. As it is indicated in [Table 2], none of the plant species produced distinguishable bands with primer pairs: amm-A, amm-B and ann-B’ which were related to d-limonene synthase [Table 2]. Limonene is biologically formed from geranyl pyrophosphate (GPP) through linalyl pyrophosphate (LPP). Limonene synthases is thought to promote the cyclization of GPP into limonene and have been previously cloned from Perilla, Mentha and Abies and occurred l-enantiomer, while d-limonene synthase was cloned from Schizonepeta tenuifolia for the first time.[16] The degenerate forward primers TerpDeg1, TerpDeg2 and TerpDeg3 were designed on the basis of the conserved sequences F (RK)(LI) LRQ (HE) G, E (GD) E (DHS)(TI) L and DD (VI)(YF) D (VI)(YF) G. PCRs using these primers with reverse primer TerpDeg4 resulted in presence of the mentioned sequences in three Labiatae species employed in this study [Table 2]. The results were supported by further PCRs using LaLIMS (forward) and GR3’ (reversed) primers, which revealed that limonene synthase should be expressed in all species except N. cataria. On the other hand, volatile oils of L. angustifolia, H. officinalis and S. sclarea contained limonene and its derivatives [Table 3].

Table 2

The results obtained from gel electrophoresis of various PCR products (cDNA of N. cataria, L. angustifolia, H. officinalis and S. sclarea) with different primer pairs

Table 3

Aroma profile composition of L. angustifolia, N. cataria H. officinalis S. sclarea young leaves obtained by HS/SPME

Interestingly, PCRs with LaLINS (forward) and GR3’Nested (reversed) primers resulted in distinguishable bands around 1800 bp in three cDNA samples, which could be related to linalool synthase, well known as a “dead end product” in the general monoterpene biosynthetic pathway.[21] Because linalool is found in the essential oils [Table 3] of three species (L. angustifolia, H. officinalis and S. sclarea), linalool synthase should be expressed simultaneously in these plants whose main constituents harbor linalool structure.

Among the selected Labiatae plants, N. cataria did not show neither limonene nor linalool synthases. The essential oil of this plant was also enriched of geraniol derivatives supporting positive bands with ddmot3 and amm primers.

The results of the head space SPME-GC-MS analysis of the aroma profiles of the above-mentioned plants have been summarized in Table 3 and showed that linalool (31.0%), linalyl acetate (18.2%), lavandulyl acetate (10.7%) and alpha-terpineol (6.3%) were found as the major compounds of L. angustifolia, while β- citronellol (8.0%), geraniol (5.5%), nerol (34.0%) and α- citral (52.0%) were identified as the main compounds of the N. cataria [Table 1]. The major components of H. officinalis and S. sclarea oils were determined as cis-pinocamphone (57.3%), β-pinene (7.0%), terpinen-4-ol (7.0%), and linalool (19.0%), linalyl acetate (51.5%), α-pinene (4.0%), respectively.

The profiles of the main components of these plants are in agreement with those reported in the literature.[6222324] Although H. officinalis was rich of cyclic monoterpenes, L. angustifolia, N. cataria and S. sclarea showed considerable amount of linear monoterpenes. The aroma profile of the above mentioned plants contained low concentration of sesquiterpenes except N. cataria, which indicated no sesquiterpene.

CONCLUSIONS

Plants of Lamiaceae family are well-known for application in traditional medicine and phytotherapy.[25] In this study, presence of linalool and limonene synthases, in four species of Labiatae family including N. cataria, L. angustifolia, H. officinalis and S. sclarea has been determined by molecular biological techniques together with the Head space SPME - GC-MS analysis of the aroma profile of these species. Taking together, H. officinalis was rich of cyclic monoterpenes, L. angustifolia, N. cataria and S. sclarea showed considerable amount of linear monoterpenes. The aroma profile of the above mentioned plants contained low concentration of sesquiterpenes except N. cataria, which indicated no sesquiterpene. The profiles of the main components of these plants are in agreement with molecular assays.