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SHORT COMMUNICATION
Photosynthetic response of Cannabis sativa L., an important
medicinal plant, to elevated levels of CO
2
Suman Chandra &Hemant Lata &Ikhlas A. Khan &
Mahmoud A. ElSohly
Published online: 25 May 2011
#Prof. H.S. Srivastava Foundation for Science and Society 2011
Abstract The effect of elevated CO
2
concentrations (545
and 700 μmol mol
−1
) on gas exchange and stomatal
response of four high Δ
9
-THC yielding varieties of
Cannabis sativa (HPM, K2, MX and W1) was studied to
assess their response to the rising atmospheric CO
2
concentration. In general, elevated CO
2
concentration
(700 μmol mol
−1
)significantly(p< 0.05) stimulated net
photosynthesis (P
N
), water use efficiency (WUE)and
internal CO
2
concentration (C
i
), and suppressed transpiration
(E) and stomatal conductance (g
s
) as compared to the
ambient CO
2
concentration (390 μmol mol
−1
) in all the
varieties whereas, the effect of 545 μmol mol
−1
CO
2
concentration was found insignificant (p<0.05) on these
parameters in most of the cases. No significant changes (p<
0.05) in the ratio of internal to the ambient CO
2
concentra-
tion (C
i
/C
a
) was observed in these varieties under both the
elevated CO
2
concentrations (545 and 700 μmol mol
−1
). An
average increase of about 48 %, 45 %, 44 % and 38 % in P
N
and, about 177 %, 157 %, 191 % and 182 % in WUE was
observed due to elevated CO
2
(700 μmol mol
−1
)as
compared to ambient CO
2
concentration in HPM, K2, MX
and W1 varieties, respectively. The higher WUE under
elevated CO
2
conditions in Cannabis sativa, primarily
because of decreased stomatal conductance and subsequently
the transpiration rate, may enable this species to survive
under expected harsh greenhouse effects including elevated
CO
2
concentration and drought conditions. The higher P
N
,
WUE and nearly constant C
i
/C
a
ratio under elevated CO
2
concentrations in this species reflect a close coordination
between its stomatal and mesophyll functions.
Keywords Cannabis sativa .Cannabaceae .Elevated CO
2
.
Photosynthesis
Abbreviations
C
a
Ambient CO
2
Concentration
C
i
Intercellular CO
2
Concentration
ETranspiration
g
s
Stomatal Conductance
P
N
Net Photosynthesis Rate
WUE Water Use Efficiency
Introduction
Increasing atmospheric CO
2
concentration and global warm-
ing are of major environmental concern around the world.
The concentration of CO
2
in the atmosphere has increased
by more than 30 %, from about 280 μmol mol
−1
in the
eighteenth century to the present level of 390 μmol mol
−1
(Mauna Loa Observatory-MLO, Hawaii), and is projected to
reach as high as 700 μmol mol
−1
by the end of the twenty
first century (Houghton et al. 1996). Thus, the future
environment will be characterized by elevated CO
2
,higher
temperature and drier climate in certain parts of the globe.
The increase in CO
2
concentration may have considerable
S. Chandra (*):H. Lata :I. A. Khan :M. A. ElSohly
National Center for Natural Product Research,
Research Institute of Pharmaceutical Sciences,
School of Pharmacy, University of Mississippi,
University, MS 38677, USA
e-mail: suman@olemiss.edu
I. A. Khan
Department of Pharmacognosy,
School of Pharmacy, University of Mississippi,
University, MS 38677, USA
M. A. ElSohly
Department of Pharmaceutics, School of Pharmacy,
University of Mississippi,
University, MS 38677, USA
Physiol Mol Biol Plants (July–September 2011) 17(3):291–295
DOI 10.1007/s12298-011-0066-6
direct and indirect effects on various life forms. Particular
effects are related to the distribution, abundance and
productivity of vegetation (Pounds and Puschendorf 2004).
Therefore, understanding the effects of the increasing
atmospheric CO
2
concentration on plants and vegetation
has become an important issue. A large number of studies
have shown enhancement in growth of the plants subjected
to both short and long-term CO
2
exposure (Kimball 1983;
Cure and Acock 1986)byaffectinganumberofbasic
physiological processes, particularly photosynthesis and
other gas exchange parameters (Ceulemans et al. 1995). A
close correlation between net photosynthesis (P
N
) and crop
yield has been reported since more than 90 % of the dry
matter of live plants is derived from photosynthetic CO
2
assimilation (Zelitch 1975). However, the magnitude of the
enhancement in photosynthesis and growth of the plants
appear to be species and genotype/variety specific (Minorsky
2002). Doubling in CO
2
concentration has been reported to
increase the yield by 30 % or more in many crops (Poorter
1993). On the other hand, inhibition of photosynthesis with
increasing CO
2
concentration has also been reported in many
plant species (Bazzaz and Garbutt 1988;Juurola2003).
Cannabis sativa L. (Cannabaceae) is a widely distributed
plant around the world. It has a long history of medicinal use
as far back as the 6th century B.C. Cannabis sativa is the
natural source of the cannabinoids, a unique group of
terpeno-phenolic compounds that accumulate in the glandu-
lar trichomes of the plant. Δ
9
-Tetrahydrocannabinolic acid
(Δ
9
-THCA) is the major cannabinoid which upon decar-
boxylation with age or heating gives rise to Δ
9
-THC, the
primary psychoactive agent (Pertwee 2006). The pharmaco-
logic and therapeutic potency of Cannabis preparations and
Δ
9
-THC have been extensively reviewed (Grinspoon and
Bakalar 1993;Mattesetal.1994 and Brenneisen et al.
1996). Despite of its medicinal importance and widespread
occurrence, to the best of our knowledge, no information
(except a previous note by our group on—one Cannabis
variety, Chandra et al. 2008) is available on the consequen-
ces of rising atmospheric CO
2
concentration on its photo-
synthesis and growth performance. This study describes the
shorttermeffectofelevatedCO
2
on photosynthetic
characteristics and stomatal response in four different high
Δ
9
-THC yielding varieties of Cannabis sativa.
Materials and methods
Plant material
Plants of four drug-type varieties of C. sativa, namely HPM
(High Potency Mexican Variety, seeds originally acquired
from Mexico), K2, MX and W1 (all from Switzerland),
were grown from seeds in the climate controlled indoor
growing facility (16 m length×6 m width) at the University
of Mississippi, USA. Since it is the female plants of this
species that are medicinally used (higher concentration of
THC and higher biomass), male plants were removed after
onset of flowering and only female plants were kept for the
experiment. Five female plants from each variety were
selected and five cuttings were made from each plant for
the photosynthetic study. Throughout the study, all the
plants were kept under strict controlled environmental
conditions (25±3 °C temperature and 55 ±5 % RH). Indoor
light (18 h photoperiod, ~700±24 μmol m
−2
s
−1
at plant
canopy level, measured by LI-COR quantum meter, model
LI-189, Lincoln, Nebraska, USA) was provided with seven
full spectrum 1000 watt HID (high intensity discharge)
lamps in combination with seven 1000 watt high pressure
sodium bulbs (Sun Systems, CA), hung above plants and
covering 110 square meter area. A hot air suction fan was
attached to each light. The bulbs were kept at least three to
four feet from the plants to avoid heating caused by the HID
bulbs. All the plants were grown in the equal size plastic pots
(30 cm diameter×28 cm height) containing 1:1:1 ratio of top
soil, sand and manure and were watered equally and regularly
to maintain identical growth condition. Out of the 25 cuttings
of each variety, 5 healthy and well established randomly
selected female clones from each variety were used for the
photosynthetic measurements and comparison.
Gas exchange measurements
To evaluate the effect of different CO
2
levels (390, 545 and
700 μmol mol
−1
) on the photosynthetic and stomatal
response of C. sativa, the gas exchange measurements
were made on the three upper undamaged, fully expanded
and healthy leaves of the five selected plants of each variety
with the help of a closed portable photosynthesis system
(Model LI-6400; LI-COR, Lincoln, Nebraska, USA).
Different levels of CO
2
were produced by portable CO
2
cylinders compatible with a LI-6400 portable photosyn-
thetic system controlled by a microprocessor. Photosyn-
thetic measurements were recorded at steady state condition
with the chamber air temperature maintained constant at
25 °C. Light (1500 μmol m
−2
s
−1
) was provided with an
artificial light source (Model LI-6400-02; light emitting
silicon diode; LI-COR), fixed above the leaf chamber and
recorded with the help of quantum sensor kept in the range
of 660–675 nm, mounted at the leaf level. The temperature
of the cuvette chamber was controlled by the integrated
Peltier coolers controlled by the microprocessor. The
CO
2
concentration supplied to the cuvette of the climatic
unit (LI-6400-01, LI-COR Inc., USA) was controlled by
mixing pure CO
2
with CO
2
free air and the CO
2
292 Physiol Mol Biol Plants (July–September 2011) 17(3):291–295
concentration was measured by infrared gas analyzer. Air
flow rate (500 μmol s
−1
) and relative humidity (55 ±3 %)
were kept nearly constant throughout the experiment.
Since steady state photosynthesis is reached within 30–
45 min (Joshi and Palni 1998; Bag et al. 2000; Joshi 2006
and Chandra et al. 2008), the leaves were kept for about
45–60 min under each set of light conditions before the
observations were recorded. Four gas exchange parameters
viz., photosynthetic rate (P
N
), transpirational water loss
(E), stomatal conductance (g
s
) and intercellular CO
2
concentration (C
i
) were measured simultaneously at steady
state under controlled light and temperature conditions. To
evaluate the leaf stomatal and mesophyll photosynthetic
efficiency, the ratio of intercellular CO
2
concentration to
ambient CO
2
concentration (C
i
/C
a
) and the ratio of
intercellular CO
2
concentration to stomatal conductance
(C
i
/g
s
) were calculated, respectively. Water use efficiency
(WUE) was calculated as the ratio of the rate of
photosynthesis and transpiration.
Statistical analysis
Values presented here are the mean of 15 replicates (Five
samples per replication with a total of three replications)
with ± SE. Two-way ANOVA was performed using
SYSTAT-11 (Systat Software Inc. San Jose, CA, USA)
statistical software.
Result and discussion
There is a growing concern that human-induced climatic
change will affect the eco-physiological processes of
plant species, and hence their productivity and distribu-
tion (Sanz-Elorza et al. 2003). Further, the responsiveness
of the species to recent and past climate changes also
suggests that climate change could act as a major cause of
extinction of some plant species in the near future
(Thomas et al. 1994). The ability of plant species to grow
and to survive in a particular environment depends on
their photosynthetic capacity (Berry and Downton 1982,
Joshi et al. 2007). It has been reported that plants with
high rates of photosynthesis and WUE have the potential
to grow faster and yield more than the species with low
photosynthesis rate and WUE under fluctuating environ-
c
a
c
b
c
bc
a
d
cd
c
bc
cd
bc
a
ab ab
ab
ab
b
bc
c
cd
cd
bc
b
cd
e
cd
de
a
bc
de
bc
e
cd
d
b
a a
ab ab
b b
bc
cd cd
d
a
b b
bc
c
cd cd
cd
cd
d d d
A
B
C
D
E
Fig. 1 Effect of different CO
2
concentrations (390, 545 and
700 μmol mol
−1
) on net photosynthesis (a), stomatal conductance
(b), transpirational water loss (c), intercellular CO
2
concentration (d)
and water use efficiency (e)ofCannabis sativa varieties namely
HPM, K2, MX and W1. Values within the same panel followed by
the same letter are not significantly different at p<0.05
Physiol Mol Biol Plants (July–September 2011) 17(3):291–295 293
mental conditions (Jones 1992; Zhang et al. 1996).
Elevated atmospheric CO
2
concentration in the environ-
ment has also been reported to enhance the photosynthesis
and growth of many plant species (Kimball 1983,Cure
and Acock 1986). It is also generally suspected that the
studies on short term exposure of elevated CO
2
overesti-
mate the relative enhancements in CO
2
assimilation rates
in plants as compared to those under the long term
exposures (Sage et al. 1989). Nonetheless, short term
studies serve a key role in providing first approximation
and indication of plants behavior under future environ-
mental conditions (Joshi 2006). Figure 1ashowstheeffect
of ambient and elevated CO
2
concentrations on net
photosynthesis of different varieties of C. sativa.Inall
the varieties, a slight but statistically insignificant (p<
0.05) increase in P
N
was observed when measurements
were made at 545 μmol mol
−1
CO
2
concentration as
compared to ambient (390 μmol mol
−1
)CO
2
, whereas, a
significant increase (p<0.05) of about 48 %, 45 %, 44 % and
38 % in P
N
was observed when the measurements were
made at 700 μmol mol
−1
CO
2
concentration as compared to
ambient level in HPM, K2, MX and W1 varieties,
respectively. The increase in P
N
due to the short term
increase in CO
2
concentration is reported to be primarily
because of an increase in carboxylation efficiency and
secondarily, due to the reduction in photorespiration (Minorsky
2002). However, the magnitude of enhancement was
reported to be species and genotype/variety specific. It
hasalsobeenreportedthat doubling of ambient CO
2
concentration increases P
N
in the plant species whose
photosynthesis is not saturated by the present ambient CO
2
level (Joshi 2006). Since photosynthesis of C3 plant
species is not saturated at the present ambient CO
2
concentration, these plants are thus reported to benefit
more than C4 plants (Bowes 1993; Joshi 2006). Average
increase of about 33 % in the rate of photosynthesis and
productivity of C3 plants has been reported with doubling
of atmospheric CO
2
concentration (Kimball 1983; Bazzaz
and Garbutt 1988; Cure and Acock 1986, Joshi 2006). In
the present investigation, under elevated CO
2
concentra-
tion (700 μmol mol
−1
) the photosynthetic rate of Cannabis
sativa was ~ 44 % higher relative to that under ambient
CO
2
(390 μmol mol
−1
).
Increasing CO
2
concentration decreased the stomatal
conductance (Fig. 1b) and subsequently, the transpiration rate
(Fig. 1c), thereby increasing the water use efficiency (Fig. 1e)
of C. sativa. Under elevated CO
2
(545 μmol mol
−1
), about
22 %, 12 %, 19 % and 13 % decrease in g
s
,19%,27%,
29 % and 32 % decrease in Eand, 36 %, 63 %, 62 % and
78 % increase in WUE was observed in HPM, K2, MX
and W1 varieties, respectively, as compared to those under
ambient CO
2
(390 μmol mol
−1
); whereas, under 700 μmol
mol
−1
CO
2
concentration, about 44 %, 43 %, 30 % and
23 % decrease in g
s
,45%,44%,50%and51%decrease
in Eand, 177 %, 157 %, 191 % and 182 % increase in
WUE was observed in HPM, K2, MX and W1 respective-
ly, as compared to those under ambient CO
2
level. It is
important to mention that compared to the report
presented earlier by Chandra et al. (2008), in the present
study differences were observed in P
N
and WUE of HPM
variety under the ambient CO
2
concentration. These
differences could be attributed to the growth stages of
plants at the time of observations recorded. However, the
effect of doubling of CO
2
on these parameters was found
comparable in both the studies. In the present study, the
observed decrease in stomatal conductance and the
reduction in the transpirational water loss under elevated
CO
2
concentration has been previously reported in many
plant species (Eamus et al. 1993;Thomasetal.1994).
Elevated CO
2
hasalsobeenreportedtoimprovethewater
use efficiency of plants (Morison 1993; Joshi 2006;
Chandra et al. 2008). The increase in C
i
(Fig. 1d), ratio
for C
i
and g
s
(C
i
/g
s
) which reflects the mesophyll
efficiency of plants and no change in C
i
/C
a
ratio (Table 1)
in response to the elevated CO
2
levels were observed in
Cannabis sativa.ConstantC
i
/C
a
values under the different
environmental conditions represent a stress free state and a
close coordination between stomatal and mesophyll
functions regulating CO
2
uptake and water loss in plants
(Jarvis et al. 1999). A similar result has been reported for
many C3 and C4 plant species exposed to elevated CO
2
levels (Morison 1993).
Parameters CO
2
Levels (μmol mol
−1
)Cannabis varieties
HPM K2 MX W1
C
i
/C
a
390 0.58± 0.06
a
0.58± 0.07
a
0.57± 0.04
a
0.60± 0.08
a
545 0.66± 0.05
a
0.59± 0.05
a
0.61± 0.06
a
0.57± 0.05
a
700 0.64± 0.08
a
0.56± 0.08
a
0.57± 0.05
a
0.61± 0.07
a
C
i
/g
s
390 1.12± 0.14
a
1.02± 0.15
a
1.22± 0.18
a
1.03± 0.16
a
545 2.19± 0.19
ab
1.62± 0.13
ab
2.31± 0.27
ab
1.15± 0.13
ab
700 3.84± 0.22
b
3.36± 0.26
b
3.15± 0.25
b
2.48± 0.21
ab
Table 1 Effect of different CO
2
concentrations on C
i
/C
a
and C
i
/g
s
ratios in Cannabis sativa varie-
ties. Values within a parameter
(C
i
/C
a
or C
i
/g
s
)followedbythe
same letter are not significantly
different at p<0.05.
C
i
—intercellular CO
2
concentra-
tion, C
a
—ambient CO
2
concentration, g
s
—stomatal
conductance
294 Physiol Mol Biol Plants (July–September 2011) 17(3):291–295
In conclusion, the effects of elevated CO
2
concentrations
on Cannabis sativa varieties were significant, leading to an
increase in P
N
and WUE of this species. However, the
magnitude of the increase was ‘variety-specific’.The
decreases in g
s
and E, and a constant C
i
/C
a
ratio under the
elevated CO
2
concentrations are the indicators of survival
potential of this species under the climatic changes leading
to drought conditions in the future.
Acknowledgements This work was supported in part by the
National Institute on Drug Abuse (NIDA), National Institute of Health
(NIH), Department of Health and Human Services, USA, Contract No.
N01DA-10-7773.
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