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Journal of Essential Oil Bearing Plants

ISSN: 0972-060X (Print) 0976-5026 (Online) Journal homepage: https://www.tandfonline.com/loi/teop20

Effects of Ethephon on Terpenoids in Cannabis


sativa L. in Vegetative Stage

Hakimeh Mansouri, Fatemeh Salari, Zahra Asrar & Fatemeh Nasibi

To cite this article: Hakimeh Mansouri, Fatemeh Salari, Zahra Asrar & Fatemeh Nasibi (2016)
Effects of Ethephon on Terpenoids in Cannabis sativa L. in Vegetative Stage, Journal of Essential
Oil Bearing Plants, 19:1, 94-102, DOI: 10.1080/0972060X.2015.1004122

To link to this article: https://doi.org/10.1080/0972060X.2015.1004122

Published online: 07 Mar 2016.

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https://www.tandfonline.com/action/journalInformation?journalCode=teop20
TEOP 19 (1) 2016 pp 94 - 102 94
ISSN Print: 0972-060X
ISSN Online: 0976-5026

Effects of Ethephon on Terpenoids in Cannabis sativa L. in Vegetative Stage

Hakimeh Mansouri*, Fatemeh Salari, Zahra Asrar, Fatemeh Nasibi


Department of Biology, Faculty of Science, Shahid Bahonar University of Kerman, Kerman, Iran
Received 01 September 2014; accepted in revised form 31 December 2014

Abstract: In the present study we investigated the effect of ethephon on changes in the amount of
many terpenoid compounds including, tetrahydrocannabinol (THC), cannabidiol (CBD), chlorophyll,
carotenoids, α-tocopherol and pyruvate as one of substrate in plastidic terpenoids biosynthesis in Cannabis
sativa at vegetative stage. Treatment with 1 and 100 μM ethephon increased THC content up to 9 fold in
vegetative stage. Plants treated with 10 μM ethephon had more CBD content in comparison with control
plants. The amount of CBD decreased in plants subjected to 5 and 100 μM ethephon. Leave area in plant
treated with ethephon did not show significant changes in comparison to control plants. Ethephon treatment
caused a declined in the amount of α-tocopherol. The levels of chlorophylls and carotenoids raised in treated
plants with 1 μM ethephon. With regard to the fact that ethephon had not negative effect on the surface of
leaves and chlorophyll content thus its application can be a suitable way for increasing of THC and α-
tocopherol in Cannabis sativa L.

Key words: Ethephon, terpenoid, cannabis, tetrahydrocannabinol (THC), cannabidiol (CBD),


α-tocopherol.

Abbreviations serve in communication and defense, for example,


THC Δ9-Tetrahydrocannabinol as attractants for pollinators and seed dispersers,
CBD Cannabidiol competitive phytoxins, antibiotics, and herbivore
MEP 2-Methyl-D-erythritol-4-phosphate repellents and toxins. Members of the terpenoid
MVA Mevalonate group also include industrially useful polymers
IPP isopenthenylpyrophosphate (rubber, chicle) and a number of pharmaceuticals
(artemisinin, taxol) and agrochemicals (pyrethrins,
Introduction azadirachtin) 16 . Isoprenoids are derived by
The terpenoids constitute the largest family of consecutive condensations of five carbon pre-
natural products and play diverse functional roles cursors, isopentenyl diphosphate (IPP) and its
in plants as hormones (gibberellins, abscisic acid), isomer dimethylallyl diphosphate (DMAPP). In
photosynthetic pigments (phytol, carotenoids), plants two distinct pathways synthesize IPP. The
electron carriers (ubiquinone, plastoquinone), acetate/mevalonate (MVA) pathway which is
mediators of polysaccharide assembly (polyprenyl shared with animals and fungi occurs in the
phosphates), and structural components of cytoplasm where sesquiterpenes (C15) and tri-
membranes (phytosterols). In addition to these terpenes (C30) such as sterols are produced. The
universal physiological, metabolic, and structural more recently identified MEP pathway (2C-
functions, many specific terpenoid compounds methyl-D-erythritol 4-phosphate) occurs in
(commonly in the C10, C15, and C20 families) plastids and is also found in protozoa, most

*Corresponding authors (Hakimeh Mansouri)


E-mail: < h_mansuori@yahoo.com > © 2016, Har Krishan Bhalla & Sons
Hakimeh Mansouri et al., / TEOP 19 (1) 2016 94 - 102 95
bacteria, and algae 8. perlite under controlled conditions (a photoperiod
Cannabis plants are interesting to human society of 12 h, Day and night temperatures were 22oC
in more ways. As a fiber plant, cannabis produces and 18oC, respectively). The plants were fertilized
some of the best and most durable fiber of natural regularly with a Hoagland’s nutrient solution every
origin. The oil of hemp seed was found to be well three days.
balanced in regards to the ratio of omega-3 and 6
fatty acids for human nutrition 10 and it is ideal as Ethephon treatment of plants
an ingredient for body oils and lipid-enriched Cannabis plants were treated when they had
creams 19. The medicinal use of cannabis has a seven pairs of leaves. Ethephon treatment done
very long history 10. by spraying the whole plants with 1, 5, 10 and
Cannabis seems a virtual factory for the pro- 100 μM ethephon solutions. Control plants
duction of secondary metabolic compounds. A sprayed with tap water. The treatment took place
variety of alkanes have been identified as well as with eight times spraying at three days intervals.
nitrogenous compounds, flavonoids and other
miscellaneous compounds. Terpenes appear in Sample preparation for cannabinoids measure-
abundance and contribute to the characteristic odor ment
of the plant. The compounds which comprise the The third leaves pair were collected and used
active drug ingredients are apparently unique to for cannabinoid measurement. Samples were dried
this genus and are termed cannabinoids. Canna- at room temperature in darkness. Sample material
binoids were originally thought to exist as the (100 mg) was placed in a test tube with 1 ml
phenolic compounds 18. Cannabis classified as chloroform. Sonication was applied for 15 min.
fiber-type and drug-type. The main difference After filtration, the solvent was evaporated to
between these two is found in the content of the dryness and the residue was dissolved in 0.5 ml
psychotropically active component, Δ9-tetrahydro- methanol.
cannabinol (THC) and its acidic precursor Chromatographic separation of cannabinoids
tetrahydrocannabinolic acid (THCA): a high was performed as descript by Rustichelli et. al.,
22
content of THC+THCA classifies as drug-type . Cannabinoid peaks were identified by cannabi-
cannabis. Fiber-type cannabis contains component noids standards (THC and CBD) that were a
that has been found to be biologically active, most generous gift from Pr. Jun Szopa Wroclaw
notably the cannabinoid cannabidiol (CBD) 9. University, Wroclaw, Poland.
Ethylene is an unsaturated hydrocarbon that
controls many of physiological processes in plant Chlorophyll and carotenoid determination
28
. Ethylene controls some processes such as Chlorophyll and carotenoids were extracted from
germination 13, growth 28, photosynthesis 30 and leaves with 80 % acetone and quantified by
falling 6. Also, ethylene involves in the plant measuring the absorbance at 663.2, 646.8 and 470
response to biotic and abiotic stress such as nm as described by Lichtenthaler, 15.
mechanical wounding, chemicals and metals,
drought, extreme temperatures, and pathogen Tocopherol extraction and chromatographic
infection 11,12. On the other hand, terpenoids play conditions
important roles in plant-plant, plant-environment Tocopherols were extracted by grinding and
and plant-animal interactions. Thus in this study homogenizing 25 mg of freeze-dried leaves
we investigate the effect of ethephon as an ethylene material in 100 μl 100 % methanol. After 20 min
donor on cannabinoids and terpenoids in cannabis of incubation at 30oC, the samples were centri-
plants at vegetative stage. fuged at 14,000 rpm for 15 min, the supernatant
was transferred to new tubes, and the pellet was
Materials and methods re-extracted twice with 250 μl 100 % methanol at
Plant material 30oC for 30 min, pooling all supernatants.
Cannabis (Cannabis sativa L.) plants were HPLC analysis was performed with liquid
grown from seed in pots (15 cm i.d, filled with chromatography equipped with a Fluorescence
Hakimeh Mansouri et al., / TEOP 19 (1) 2016 94 - 102 96
detector. C18 was used for separation. Methanol Fiber study
(100 %) was used as mobile phase; the flow rate The cross section pieces from third internod were
was 1 ml min -1. Fluorescence detector was cut with microtome. They placed on the distilled
accomplished at 295-335 nm for α-tocopherol 23. water until microscopic survey. The cuts were
Injection volume was 10 μl. The identities of α- analyzed under light microscope (company
tocopherol in plant samples were determined by Olympus, Provis AX 70). In overview (40-fold
comparing with (±)-α-tocopherol standard was enlargement) we qualitatively studied the changes
purchased from Sigma (Germany). in primary fibers in the cross section of the stems.

Determination of pyruvate of chloroplast Data analysis


Extraction of chloroplast Data were subjected to statistical analysis using
In order to measurement the amount of pyruvate statistical software package SPSS version 14. One
in chloroplasts, we have to extract chloroplasts way analysis of variance (ANOVA) followed by
from leaves tissue. First, the large vein was Duncan multiple range test were employed and
isolated. 0.5 g of leaves with 3 ml of Tris-sucrose the differences between individual means were
buffer (0.3 M sucrose, 0.2 M tris HCL, 5 mM deemed to be significant at p<0.05. The values
MgSo4) was pulverized. The resulting solution was are ±SD for three samples in each group.
filtered and for 5 min was centrifuged (1000 rpm).
Supernatant was removed and residues were Result
dissolved in 2 ml of Tris-sucrose buffer. The diluted Effect of ethephon on cannabinoid content
suspension of chloroplasts was maintained for Our results showed that THC and CBD contents
measurement of pyruvate by Anthon and Barrett were equal at leaves (Fig. 1). Treatment with
method 3. ethephon increased THC content. In this regard
the lowest and highest concentration of ethephon
Measurement of leaves area had stronger effect. The leaves THC content in
For measuring leaves area, we made paper these treatments was 8.9 fold of untreated plants.
copies of the third leaves. Then the weight of However, treatment with 5 and 10 μM ethephon
square 1*1 of paper was measured. Based on the increased THC content in leaves but it was not
proportional relationship between leaves weight, considerably. The amount of CBD in plants treated
its surface was calculated. with 1, 5 and 100 μM ethephon decreased in com-
Cannabinoid content (mg g-1 dw)

μM)
Ethephon (μ
Fig. 1. The effects of ethephon on THC and CBD content of leaves in cannabis plants.
Values are means of three replications ± SD. Asterisks indicate the significance
of difference at P< 0.05 level according to Duncan’s multiple range test
Hakimeh Mansouri et al., / TEOP 19 (1) 2016 94 - 102 97
parison to control but treatment with 10 μM ethe- The amount of chlorophyll a, b and total
phon increased CBD content up to 4 fold (Fig. 1). increased in 1 μM ethephon treatment. In other
ethephon concentrations, the changes in chloro-
Effect of ethephon on chlorophyll and caroten- phyll content were not significant in comparison
oids content to control plants (Fig. 2).
Since ethylene may function as major switch The changes in carotenoids content in plant
between growth and defense and the associated treated with ethephon were similar to those of
changes in resource allocations; we investigated chlorophyll content (Fig 3). Treatment with 1 μM
this probably relationship between plastidic ethephon stimulated carotenoid accumulation on
primary and secondary terpenoids with chloro- leaves, but 5, 10 and 100 μM ethephon treatments
phyll, carotenoid and α-tocopherol measurement. were not affective in this regard.
Chlorophyll content (mg g-1 dw)

Ethephon (μ μM)
Fig. 2. The effects of ethephon on chlorophyll content of leaves in cannabis plants.
Values are means of three replications ± SD. Asterisks indicate the significance
of difference at P< 0.05 level according to Duncan’s multiple range test
Carotenoids content (mg g-1 dw)

Ethephon (μμM)
Fig. 3. The effects of ethephon on carotenoids content of leaves in cannabis plants.
Values are means of three replications ± SD. Asterisks indicate the significance
of difference at P< 0.05 level according to Duncan’s multiple range test
Hakimeh Mansouri et al., / TEOP 19 (1) 2016 94 - 102 98
Effect of ethephon on á-tocopherol content Effect of ethephon on leaves area
The amount of α-tocopherol in treated plants Ethephon treatment with this method and
with 5, 10 and 100 μM ethephon was lower than concentration did not effect on decreasing of
control plants. Treatment with 1 μM ethephon did leaves area of plants in comparison to control
not effect on α-tocopherol (Fig. 4). plants (Fig. 6).

Effect of ethephon on pyruvate Effect of ethephon on primary fibers in stem


Control plants had the highest pyruvate content. The amount of the individual layers in the cross
Pyruvate content of plants treated with ethephon section of the stem and the number of fiber cells
was lower than the control plants (Fig. 5). per layer increased in 1 μM ethephon treatment
μg g-1 dw)
α-Tocopherol content (μ

μM)
Ethephon (μ
Fig. 4. The effects of ethephon on α-tocopherol content of leaves in cannabis plants.
Values are means of three replications ± SD. Asterisks indicate the significance
of difference at P< 0.05 level according to Duncan’s multiple range test
Pyruvate content (mg g-1 dw)

Ethephon (μμM)
Fig. 5. The effects of ethephon on pyruvate content of leaves in cannabis plants.
Values are means of three replications ± SD. Asterisks indicate the significance
of difference at P< 0.05 level according to Duncan’s multiple range test
Hakimeh Mansouri et al., / TEOP 19 (1) 2016 94 - 102 99

leaf area (cm2)

Ethephon (μ μM)
Fig. 6. The effects of ethephon on leaves area in cannabis plants. Values are means
of three replications ± SD. Asterisks indicate the significance of difference
at P< 0.05 level according to Duncan’s multiple range test
also cell wall diameter had been increased in 1 were amorphous; the wall between the fiber cells
μM ethephon treatment. But in other treatments was separated in this treatment (Fig. 7).
(5, 10 and 100 μM) area of primary fiber cells
and their cell lumen was increased. In 100 μM Discussion
ethephon treatment, the fiber and cambium cells In this study, we were evaluated the effect of

Fig. 7. The effect of ethephon on fiber cells on the cross section of stem third internod
in cannabis plants. (40 fold enlargement). a) control, b) 1 μM ethephon, c)
5 μM ethephon, d) 10 μM ethephon, e) 100 μM ethephon.
Hakimeh Mansouri et al., / TEOP 19 (1) 2016 94 - 102 100
ethylene on some primary and secondary terpen- ethephon can suitable treatment to increase
oids compounds in Cannabis sativa L. The mount cannabinoids in cannabis plants.
of THC increased in all ethephon treatments but Carotenoids content enhanced in the lowest
the mount of CBD increased only by 10 μM ethe- ethephon concentration (1 μM) similar to chloro-
phon treatment and other treatments decreased the phyll responds. In coincidence with our results,
mount of CBD. Concurrent with our results Bate ethylene induced carotenoid content in Kinnow
et. al., 4 reported that ethylene stimulates synthesis mandarin peels 17 and fig fruits 20 but Alexieva et.
of ginsenoside in ginseng. Ethylene is known to al., 1 reported a decrease in carotenoid content on
enhance the production of terpenoid phytoalexins detached leaves of Arabidopsis thaliana.
in tobacco cultures 31. Also considerable increases The amount of α-tocopherol decreased by
in the levels of notkaton (sesquiterpene) were ethephon treatment. Tocopherols are involved in
observed in the picked and unpicked grapefruits protecting lipids and the photosynthetic apparatus
treated with ethylene. Tomas et. al., 29 suggested against created oxidative damage by variety of
that ethylene regulates nootkatone biosynthesis by biotic and abiotic stresses. In cannabis tetrahydro-
accelerating the maturation-senescence processes cannabinolic acid (THCA) is biosynthesized from
in grapefruit rind. El-Keltawi and Croteau 7 cannabigerolic acid (CBGA). This reaction
reported the influence of ethephon on the consti- depends on molecular oxygen and produces
tuents of monoterpenes of Mentha piperita. Most hydrogen peroxide and THCA at a molar ratio of
of the growth hormone studies on M. piperita or 1:1 (CBGA + O2JTHCA + H2O2) 27. Probably,
M. arvensis impact on the enzymes of biosynthetic THCA biosynthesis reaction can act as a defense
pathways 26. mechanism by using molecular oxygen instead of
The phytyl (C20) conjugates such as chloro- α-tocopherol in this condition.
phylls and tocopherols, and carotenoids (C40) are Pyruvate and glyceraldehydes-3-phosphate are
produced by MEP pathway in chloroplast. In order precursors of IIP biosynthesis in MEP pathway.
to investigation the ethephon effects on the MEP In this work, ethephon treatment resulted in
pathway regulation, we measured the main end decrease in pyruvate content that this may be due
products of this pathway in ethephon-treated to the role of ethephon on stimulation of bio-
plants. Ethephon treatment resulted in increase in synthesis pathway of plastidic terpenoids.
chlorophylls and carotenoids content. An increase Our result showed that the lowest concentration
in chlorophyll content has also detected in of ethephon (1 μM) probably has a beneficial effect
cucumber cotyledons 2, Sugarcane 14, carnation on primary fiber cells. This treatment increased
flowers 5. Also many reports showed that an the cell wall diameter. Of course to confirm this
exogenous application of ethylene to green plant subject we need to quantitative analysis of fibers.
materials induced a decrease in chlorophyll con- Also Shi et. al., 25 reported a major role for ethylene
tent 21,9. However, we think in these works high in cotton fiber cell elongation. They suggested that
concentration of ethylene or longer time of ethylene may promote cell elon-gation by
treatment was used in comparison with our increasing the expression of sucrose synthase,
experiment. Because ethephon did not effect on tubulin, and expansin genes.
decreasing of leaves area and photosynthetic We concluded that probably low concentrations
pigments, it can be supposed that these ethephon of ethephon can use either for increasing canna-
concentrations and treatment method could not binoids or fiber quality in Cannabis sativa without
inhibit plant growth. Thus low concentrations of to have adverse effect on plant growth.

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