Caffeine Extraction, Enzymatic Activity and Gene Expression of Caffeine Synthase from Plant Cell Suspensions

Summary

This protocol describes an efficient methodology for the extraction and quantification of caffeine in cell suspensions of C. arabica L. and an experimental process for evaluating the enzymatic activity of caffeine synthase with the expression level of the gene that encodes this enzyme.

Abstract

Caffeine (1,3,7-trimethylxanthine) is a purine alkaloid present in popular drinks such as coffee and tea. This secondary metabolite is regarded as a chemical defense because it has antimicrobial activity and is considered a natural insecticide. Caffeine can also produce negative allelopathic effects that prevent the growth of surrounding plants. In addition, people around the world consume caffeine for its analgesic and stimulatory effects. Due to interest in the technological applications of caffeine, research on the biosynthetic pathway of this compound has grown. These studies have primarily focused on understanding the biochemical and molecular mechanisms that regulate the biosynthesis of caffeine. In vitro tissue culture has become a useful system for studying this biosynthetic pathway. This article will describe a step-by-step protocol for the quantification of caffeine and for measuring the transcript levels of the gene (CCS1) encoding caffeine synthase (CS) in cell suspensions of C. arabica L. as well as its activity.

Introduction

Caffeine is a secondary metabolite that is biosynthesized by plants of the genus Coffea¹. This alkaloid belongs to the methylxanthine family and is regarded as a chemical plant defense because it can act against the adverse effects of pathogens and herbivores^2,3. In addition, this metabolite is responsible for the stimulating properties of the coffee drink, which is commonly consumed worldwide^4,5. Due to its properties, several research groups are interested in studying the biosynthetic pathway and catabolism of caffeine^6,7. Currently, plant in vitro cell/tissue cultures serve as an alternative for evaluating caffeine accumulation under various biotic and abiotic strategies^8,9.

Caffeine biosynthesis involves the hydrolytic release of 7-methylxanthine from the corresponding ribose nucleoside followed by ordered N-methylations at positions 3 and 1. A specific S-adenosyl methionine (SAM)-dependent N-methyltransferase (NMT) catalyzes the methylation at position 7, whereas theobromine synthase (TS) and CS are involved in the 3- and 1-methylations, respectively, producing theobromine and caffeine. The study of genes encoding distinct NMTs has allowed understanding the mechanism that regulates caffeine production^10,11. CS, which has N-methyltransferase activity, catalyzes the last two steps of the biosynthetic pathway of caffeine¹¹. In coffee tree seedlings, it has been shown that light radiation can increase CS activity, which results in an increase in caffeine biosynthesis. Recently, we showed that the maintenance of cell suspensions of C. arabica L. under light irradiation is the optimal condition for evaluating the effects that produce abiotic stress factors impacting the biosynthetic pathway of caffeine⁸. The information obtained in these studies may have applications in metabolic engineering and systems biology for maximizing the study of the caffeine biosynthetic pathway in such in vitro systems.

Given the advantages of obtaining a suitable model for the study of caffeine biosynthesis, we optimized the extraction conditions for caffeine on cell suspensions of C. arabica L. It was also possible to develop a useful protocol for studying the enzymatic activity as well as methodological steps for assessing the level of gene transcripts of Coffea caffeine synthase 1 (CCS1) encoding this enzyme. Herein, we report a protocol to extract and quantify caffeine in C. arabica cell suspensions by thin-layer chromatography and densitometry (TLC-densitometry).

Protocol

1. Caffeine Extraction in Cell Suspensions of C. arabica L.

1. Use C. arabica cell suspensions⁹. Maintain the suspensions by biweekly subcultures in Murashige and Skoog medium at pH 4.3 with constant 100 rpm shaking at 25 °C under continuous light (8.3 W/m²).
2. Harvest the cells under vacuum filtration using 11 µm pore filter paper and a Buchner funnel.
3. Register the fresh weight of the collected cells using a scale, wrap them in aluminum foil, freeze them in liquid nitrogen and keep them at -80 °C until analysis.
4. Lyophilize the frozen cellular material for 72 h.
5. Register the weight of the lyophilized cells in order to estimate the dry weight yield.
6. Store the powdered material in reusable polyethylene bags (9 cm x 7.5 cm). Macerate the material to a fine powder and store in a desiccator until use.
7. Measure 0.5 g of dry cellular matter on the analytical scale and add it to a glass flask (25 mL).
   1. Add 10 mL of acetone to the lyophilized cells and mix in a vortex mixer for 30 s for homogenization. Then, seal the flask with aluminum foil.
   2. Mix at 100 rpm using a rotator at room temperature (25 °C) for 5 h.
8. Transfer all material to a 15 mL conical tube and centrifuge at 13,000 x g for 10 min. Transfer the liquid phase to a new tube and reduce to it dryness at room temperature in the fume hood.
9. Resuspend the sample with 25 µL of acetone.

2. Conditions for the Assessment of Caffeine by Thin Layer Chromatography (TLC)-Densitometry

1. Apply 1 µL of the previously resuspended caffeine extract to the stationary phase.
   Note: Silica gel plates (F254) are used as the stationary phase. Before applying the samples, the plates were developed with 10 mL of chloroform-methanol [9:1 v/v] in a chromatography chamber (14 cm x 12 cm x 9.5 cm), and the plates were dried at room temperature. The characteristics of the chromatographic plate where the samples are applied are shown in Figure 1. The chamber was saturated for 30 min with solvent before developing the plate. TLC plates should handle using gloves to avoid contamination.
2. Develop the TLC in the chromatography chamber with 10 mL of the mobile phase cyclohexane-acetone [40:50 v/v].
   Note: This mixture allows the separation of caffeine at a retention factor (Rf) of 0.34.
   1. Remove the TLC plate from the chamber and let it dry completely at room temperature.
3. Visualize caffeine bands in short wavelength ultraviolet light (UV, 254 nm). Use a compact UV lamp. Additionally, for this step, use goggles with UV protection.
4. Quantify caffeine levels by densitometry at 273 nm. The instrument is controlled with chromatography software.

3. Ribonucleic Acid (RNA) Isolation

1. Take the cell suspensions from step 1.1 and vacuum filtrate with filter paper (11 µm pore size).
2. Collect the cellular material from the filter paper, weigh out 0.5 g of cellular material on an analytical balance and pack it in aluminum foil. Freeze the sample with liquid N₂.
3. Macerate the cell sample in a porcelain mortar with liquid nitrogen and add 1 mL of RNA isolation reagent until homogenized.
   Note: Sterilize the porcelain mortars in a muffle furnace at 300 °C for 6 h. RNA isolation reagent contains phenol, guanidine isothiocyanate and other components.
   1. Transfer 500 µL of the sample to a sterile microcentrifuge tube (1.5 mL). Add 300 µL of chloroform-isoamyl alcohol (24:1) and 300 μL of equilibrated phenol (pH 8.0).
4. Mix the sample with a vortex mixer and then centrifuge it at 20,000 x g for 15 min at 4 °C.
5. Transfer 300 μL of the upper phase to a microcentrifuge tube. Add 200 μL of isopropanol. Incubate the tube for 1 h at -20 °C.
6. Centrifuge the tube at 12,000 x g for 10 min at 4 °C, and then decant the liquid phase.
   1. Add 1 mL of ethanol (70%) to form a pellet in the tube. Centrifuge at 12,000 x g for 10 min and decant. Repeat this step twice.
7. Dry the sample for 1.5 h at room temperature (25 °C). Resuspend the RNA extract with 25 μL of diethyl pyrocarbonate (DEPC)-treated water.
   Note: For the RNA extraction, use water treated with 0.1% DEPC v/v.

4. Incubation of RNA Transcript with Deoxyribonuclease (DNase)

1. Into a sterile microcentrifuge tube, add 2 µg of total RNA extract. Add 1 μL of 10x reaction buffer (100 mM Tris-HCl, 25 mM MgCl₂, 1 mM CaCl₂). Add 1 μL of 1 U/µL DNase.
   1. Bring the reaction to a final volume of 10 μL with DEPC-treated water. Mix the sample and centrifuge briefly at 2,000 x g for 1 min.
2. Incubate the sample at 37 °C for 30 min.
3. Stop the reaction with the addition of 1 μL of 50 mM disodium ethylenediaminetetraacetate dihydrate (Na₂EDTA) and incubate the sample at 65 °C for 10 min.
4. Analyze the integrity of the RNA by agarose gel electrophoresis.
   1. Prepare a standard 1% agarose gel and stain it with 1 µL of 3x intercalating nucleic acid stain solution.
   2. Apply 500 ng of RNA to the agarose gel and run the gel.
   3. Visualize the integrity of the RNA using a gel photo documentation system.
   4. Use RNA as the template for cDNA synthesis by reverse transcriptase.

5. Complementary Deoxyribonucleic Acid (cDNA) Synthesis

1. Add 2.5 μg of total RNA to a microcentrifuge tube. Add 1 μL of oligo-deoxythymine (dT) primer and fill the tube to a 13 μL final volume with nuclease-free water. Mix the sample and then centrifuge a 2,000 x g for 1 min.
2. Incubate the sample at 65 °C for 5 min and then at 4 °C for 2 min.
3. Add 4 μL of 5x reaction buffer used for reverse transcriptase, 2 μL of deoxynucleotide triphosphates (dNTPs) mix (10 mM of each dNTP) and 1 μL of reverse transcriptase (200 U/µL). Mix gently and centrifuge at 2,000 x g for 1 min.
4. Incubate the sample at 45 °C for 50 min and then at 70 °C for 10 min.
5. Quantify the cDNA concentration using a UV spectrophotometer.
6. Use the cDNA as the template for the polymerase chain reaction (PCR).
   Note: The reaction buffer was used as 10x (step 4.1.1) and 5x times concentrated (step 5.3), respectively.

6. Real-Time Quantitative PCR (qPCR) to Amplify the Gene CCS1

1. Add 7.5 μL of Taq DNA polymerase 2x (0.1 U/mL) and 0.1 μM of 5-carboxy-X-rhodamine (ROX) to a PCR microcentrifuge tube.
   Note: The hot start Taq DNA polymerase 2x was used as a solution 2x concentrated.
   1. Add 5 μL of nuclease-free water.
   2. Add 0.75 μL each of 10 μM forward and reverse primer to amplify the gene CCS1 (AB086414) (forward: 5’-CCTGTATCCTGCGATGAACA-3’ and reverse: 5’-AACACTATCATAGAAGCCTTTG-3’). Use primers for tubulin as the house-keeping gene in a separate reaction (forward: 5’-GTGCCCAACTGGGTTCAA-3’ and reverse: 5’-CCTTCCTCCATACCTTCACC-3’)⁹.
   3. Add 600 ng of cDNA template.
2. Perform the amplification reaction at 50 °C for 2 min and 95 °C for 5 min. Incubate the reaction in the real-time PCR system for 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s.
3. Incubate the sample at 50 °C for 30 s and 20 °C for 10 s.
4. Analyze the data with the PCR software.
5. Calculate the relative expression by the 2-^ΔΔCT method described by Livak and Schmittgen (2001)¹².
   Note: Analyze a control reaction that contains all components except the DNA template (no template control, NTC) to discard contaminated reagents or amplification primer-dimers.

7. Total Protein Extract of Cell Suspensions of C. arabica L.

1. Filter cell suspensions under vacuum with a medium-pore filter paper.
2. Weigh 1 g of cellular material and pack it in aluminum foil.
   1. Freeze the sample with liquid nitrogen. Macerate the sample in a sterilized porcelain mortar to obtain a fine powder.
3. Transfer the sample to a glass vial and add 2.5 mL of extraction buffer (400 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), at pH 8.0, containing 10 mM β-mercaptoethanol, 5 mM Na₂EDTA and 0.5% (w/v) sodium ascorbate.
   1. Mix the sample in a vortex mixer for 2 min.
      Note: It is important that the sample is kept on ice to prevent protein denaturation.
4. Centrifuge the sample at 20,000 x g for 20 min at 4 °C and transfer 500 µL aliquots into cryogenic vials (2 mL). Store samples at -72 °C until use.
5. Measure the protein concentration of the sample at 562 nm in a spectrophotometer using bicinchoninic acid assay with bovine serum albumin as the standard.

8. Enzymatic Assay for Caffeine Synthase

1. Prepare a reaction mixture in a microcentrifuge tube containing 200 µM SAM, 4.07 kBq of methyl [³H]-SAM, 200 µM theobromine, 200 µM MgCl₂, and 5 µM caffeine. Increase the volume to 200 µL with 100 mM Tris-HCl at a pH of 8.0.
2. Keep the microcentrifuge tube on ice and add a volume of the soluble fraction containing 7-9 mg of protein. Then, mix by vortex and incubate for 30 min at 30 °C in a thermostatic bath/circulator. For a batch of several samples, incubate each sample in duplicate in 1 min periods to give enough time to stop the reactions for all samples in an exact time period of 30 min.
   1. Add 1 mL of chloroform to stop the reaction and then shake the sample with a vortex mixer. Shake the sample using a vortex mixer for 3 min to remove the radioactive caffeine in the solvent.
3. Centrifuge samples at 11,000 x g for 5 min and carefully recover a volume of 900 µL. Then, transfer the samples to scintillation vials.
4. Evaporate the chloroform to complete dryness in the hood at room temperature at 25 °C. Add 5 mL of scintillation fluid to the vial and mix the sample.
5. Analyze the radioactivity incorporated into the caffeine in a scintillation counter.

Results

Caffeine extracts obtained via the process presented here were analyzed by TLC-densitometry by subjecting the samples to plate chromatography according to the scheme shown in Figure 1. To quantify the levels of caffeine in the cell extracts, a curve with various concentrations of commercial standard for this compound was used (Figure 2A). The pattern of absorbance for caffeine was analyzed in the visible light spectrum (UV-VIS) u...

Discussion

We present here the optimal conditions for evaluating the caffeine content, CS activity and transcript levels in an in vitro plant tissue culture, such as cell suspensions of C. arabica. Previous reports have confirmed that maintaining cells under light irradiation and in the presence of theobromine in the culture medium are suitable parameters for increasing the level of caffeine, making it possible to evaluate the caffeine separation methods using reversed-phase high-performance liquid chromatography ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Murashige & Skoog Basal salt mixture	PhytoTechnology Laboratories	M524	Packge Size: 50 L Reagent (mg/L) Ammonium Nitrate (1650) Boric acid (6.2) Calcium chloride, anhydrous (322.2) Cobalt Chloride•H2O (0.025) Cupric Sulfate•5H2O (0.025) Na2EDTA•2H2O (37.26) Ferrous Sulfate•7H2O (27.8) Magnesium Sulfate, Anhydrous (180.7) Manganese Sulfate•H2O (16.9) Molybdic Acid (Sodium Salt)• 2H2O (0.25) Potassium Iodide (0.83) Potassium Nitrate (1900) Potassium Phosphate, Monobasic (170) Zinc Sulfate•7H2O (8.6) Supplemented with myo-inositol (100) thiamine (10) cysteine (25) sucrose (30000) 2,4-dichlorophenoxyacetic acid (3) 6-benzylamine purine (1)
Caffeine	SIGMA	C0750-5G	STANDARD-5g
Theobromine	SIGMA	T4500	20 g
CAMAG TLC Scanner-4	CAMAG	27.62
WinCATS Planar Chromatography Manager software	CAMAG	1.4.10	Software
Isoamyl alcohol (24:1)	SIGMA	C-0549	500 mL
Cyclohexane	JALMEX	C4375-13	1 L
Acetone	J.T. BAKER	900643	4 L
Methanol	J.T. BAKER	9093-03	4 L
Chloroform	JALMEX	C-4425-15	3.5 L
TLC silica gel 60 F254	Merck	1.05554.0001	TLC plate
β-mercaptoethanol	M6250	SIGMA	100 mL
(+)-sodium L- ascorbate	A4034	SIGMA	100 g
Trizma base	SIGMA	T6066	1 Kg
Hydrochloric acid 36.5-38%	J.T. Baker	9535-05	2.5 L
Pierce BCA Protein Assay Kit	Thermo scientific	232227	Kit
Methyl [3H]-S-adenosyl methionine	Perkin Elmer	NET155	Specific activity of 15 Ci/mmol
Liquid scintillation vials	SIGMA	Z253081
Thermostatic bath/circulator	Cole Parmer	60714
Micro centrifugue tube	Eppendorf		Tube of 1.5 mL
Cryogenic vials	Heathrow Scientific	HS23202A	2 mL
Centrifuge 5804	Eppendorf	5804 000925
Vortex	Thermolyne	LR 5947
Porcelain mortar	Fisherbrand	FB961B
Filter paper	Whatman	Z274844	Porosity medium
Picofuge	Stratagene	400550	2000 x g
Analytical balance	AND	HR-120	Model HR-120
Scintillation counter	Beckman Coulter	6500
Gel photodocumentation system	Bio-Rad	Chemic XRS	Model Chemic XRS
Compact UV lamp	UVP	95002112	UVGL-25
Scienceware HDPE Buchner funnel	SIGMA	2419907	Type 37600 mixer
TRIzol reagent	Thermo scientific	15596-018	200 mL
ReverdAid Reverse transcriptase	Thermo scientific	#EP0441	10000 U
Oligo (dT)18 primer	Thermo scientific	#S0131	100 µM
DNase I, RNase-free	Thermo scientific	#EN0525	1000 U
Magnesium chloride	Thermo scientific	EN0525	1.25 mL
Ethylenediaminetetraacetic acid	Thermo scientific	EN0525	1 mL
dNTP mix	Thermo scientific	R0191	R0191
SYBR Green qPCR Master Mix (2X)	Thermo scientific	K0251	For 200 reactions of 25 µL
PikoReal	Thermo scientific	2.2	Software
Phenol, pH 8.0, equilibrated, Molecular Biology Grade, Ultrapure	USB	J75829	100 mL
Isopropyl alcohol	Karal	2040	1 L
Ethyl alcohol	SIGMA	64175	1 L
Diethyl pyrocarbonate	SIGMA	D5758	100 mL
Lab Rotator	LW Scientific	Mod. LW210