Natural sesquiterpene; Flavoring Agent

Herbivore-induced plant volatiles (HIPVs) play important ecological roles in defense against stresses. In contrast to model plants, reports on HIPV formation and function in crops are limited. Tea (Camellia sinensis) is an important crop in China. α-Farnesene is a common HIPV produced in tea plants in response to different herbivore attacks. In this study, a C. sinensis α-farnesene synthase (CsAFS) was isolated, cloned, sequenced, and functionally characterized. The CsAFS recombinant protein produced in Escherichia coli was able to transform farnesyl diphosphate (FPP) into α-farnesene and also convert geranyl diphosphate (GPP) to β-ocimene in vitro. Furthermore, transient expression analysis in Nicotiana benthamiana plants indicated that CsAFS was located in the cytoplasm and could convert FPP to α-farnesene in plants. Wounding, to simulate herbivore damage, activated jasmonic acid (JA) formation, which significantly enhanced the CsAFS expression level and α-farnesene content. This suggested that herbivore-derived wounding induced α-farnesene formation in tea leaves. Furthermore, the emitted α-farnesene might act as a signal to activate antibacterial-related factors in neighboring undamaged tea leaves. This research advances our understanding of the formation and signaling roles of common HIPVs in crops such as tea plants.[1]

Enzyme Assay and Functional Characterization of CsAFS[1]
Two different substrates, FPP and GPP, were separately reacted with CsAFS protein (15 μg) in buffer (1 mL). pET32a empty vector protein was treated under the same conditions as control. All reactions were performed in bottles sealed using parafilm. The reaction was performed at 30 °C for 1 h, and α-farnesene was collected by solid-phase microextraction (SPME) (2 cm, 50/30 μm, DVB/Carboxen/PDMS Stable Flex, Bellefonte, PA, USA) during this time. The enzyme assay buffer was composed of 10 mM Na2HPO4, 1.8 mM NaH2PO4 (pH 7.3), 140 mM NaCl, 10 mM MgCl2, 5 mM dithiothreitol, 500 mM KCl, 1 mM MnCl2, 0.05% (w/v) NaHSO3, and 10% (v/v) glycerol, adjusted to the optimum pH 7.0 [16]. Volatiles absorbed by SPME and the α-farnesene standard dissolved in CH2Cl2 were subjected to GC-MS analysis.

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Analysis of CsAFS Activity in N. Benthamiana Overexpression Lines[1]
After subcellular location analysis, the infiltrated fresh tobacco leaves were frozen with liquid nitrogen and kept at −80 °C for further study. We added 1.8 mL CH2Cl2 to extract 300 mg finely powdered fresh tea leaves followed by mixing 5 nmol ethyl decanoate as internal standard. Then all the samples were extracted at 25 °C for 5 hours. Anhydrous Na2SO4 was used to remove water in extraction solution that was then concentrated using nitrogen flow. The final 200 μL extracts and the α-farnesene standard dissolved in CH2Cl2 were then subjected to a GC-MS analysis.

α-Farnesene Treatment[1]
The plant materials picked from C. sinensis cv. Yinghong No. 9 were placed in a closed jar containing a cotton ball. α-Farnesene standard (500 µL, 4 mM) in dichloromethane (CH2Cl2) was injected into the cotton ball every 12 h as treatment. The total α-farnesene content in the treatment was 6 µmol. In the control groups, CH2Cl2 was injected into the cotton ball instead of the α-farnesene standard. The total volume of CH2Cl2 in the control was 1.5 mL. Samples were collected at 24 h and 36 h, frozen with liquid nitrogen, and kept at −80 °C for further study. As α-farnesene is water insoluble, CH2Cl2 was used as solvent to dissolve the α-farnesene standard to achieve fumigation. A previous report indicated that the influence of CH2Cl2 was the same as that of H2O, with no negative effects.

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Analysis of α-Farnesene Content in Tea Leaves[1]
In herbivores attack treatment and wounding treatment, 200 mg finely powdered fresh tea leaves of each sample was extracted with 700 μL CH2Cl2 containing 2 nmol ethyl decanoate as an internal standard. In JA treatment, we adopted 1.8 mL CH2Cl2 containing 5 nmol ethyl decanoate which was considered as internal standard, to extract 500 mg finely powdered fresh tea leaves of each sample. Afterward, all samples were shaken for 5 h at 25 °C. The filtered extraction solution was dried with anhydrous Na2SO4 to remove water and evaporated under nitrogen flow until had a 100 μL extract. Finally, samples were analyzed by GC-MS.

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Analysis of Phytohormones Content in Tea Leaves[1]
In wounding treatment and α-farnesene treatment, we used 2 mL ethyl acetate to extract 200 mg finely powdered fresh tea leaves of each sample. This was followed by vortexing for 30 s and adding [2H5] JA to the mixture as an internal standard in continuous wounding treatment, while adding [2H4] SA, [2H5] JA and [2H6] ABA to the mixture as internal standards in α-farnesene treatment. Afterward, all samples were put in ice-cold water with ultrasound for 20 min, and the supernatants were collected through centrifuging at 10,000× g for 5 min at 4 °C. Then all extraction solutions were dried under nitrogen flow, redissolved in methanol (200 µL), and filtered through a 0.22 µm membrane. Finally, samples were analyzed by an ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS).

[1]. Formation of α-Farnesene in Tea ( Camellia sinensis) Leaves Induced by Herbivore-Derived Wounding and Its Effect on Neighboring Tea Plants. Int J Mol Sci. 2019 Aug 25;20(17):4151.

Alpha-farnesene is a farnesene that is 1,3,6,10-tetraene substituted by methyl groups at positions 3, 7 and 11 respectively.
alpha-Farnesene has been reported in Camellia sinensis, Aristolochia triangularis, and other organisms with data available.
See also: Chamomile (part of); Cannabis sativa subsp. indica top (part of).

C15H24

204.35

204.188

502-61-4

113244-64-7

5281516

Colorless to light yellow liquid

0.844-0.8790 g/mL at 25ºC(lit.)

279.6ºC at 760 mmHg

<25 ℃

113.2ºC

n20/D 1.490-1.505(lit.)

5.201

0

0

6

15

270

0

C=C/C(=C/C/C=C(\C)/CCC=C(C)C)/C

CXENHBSYCFFKJS-VDQVFBMKSA-N

InChI=1S/C15H24/c1-6-14(4)10-8-12-15(5)11-7-9-13(2)3/h6,9-10,12H,1,7-8,11H2,2-5H3/b14-10+,15-12+

(3E,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene

α-Farnesene; alpha-Farnesene; Farnesene; 502-61-4; a-farnesene; (E,E)-alpha-farnesene; (3E,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene; .alpha.-Farnesene; (E)-alpha-Farnesene;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)