Transcript Slide 1

Effect of Jasmonic Acid on Biomass Accumulation and Enzyme Activity in Switchgrass
Jocelyn Bidlack, Paul Olson, Carmen Cowo, Erin Ralstin, Robert Dinger, and Jim Bidlack
University of Central Oklahoma, Edmond, OK 73034
Under conditions of osmotic stress, plants regulate stomatal closure to conserve
moisture by limiting the transpiration flux. Modification of the stomatal opening
concurrently limits the uptake of CO2, greatly affecting photosynthetic rate and
leading to the increased production of reactive oxygen species (ROS) (Sgherri et al.
1993). The ROSs initiate a cascade of reactions, resulting in the destruction of
organic macromolecules such as lipids, proteins, and nucleic acids (Shewfelt and
Purvis 1995).
Jasmonic acid (JA) is a stress-induced hormone that modulates the expression of
antioxidative defensive systems. The exogenous application of JA prior to the
initiation of osmotic stress has the potential to moderate the effects of drought (Li et
al. 1998, Rohwer and Erwin 2008, Wang 1999). Treatment effectively induces
biosynthesis and accumulation of the ROS-scavenging antioxidants (Gao et al.
2004), and induces increased activity of the ROS-scavenging enzymes.
JA application may alter the production of secondary compounds with antioxidant
properties indicated by significantly affecting the activity of hydroxymethyl glutaryl
CoA reductase-HMGR (the rate limiting enzyme for the production of terpenoids).
In addition, JA application is expected to alter the activity of phenylalanine
ammonia lyase (PAL), the rate limiting enzyme for the production of the phenolic
compounds such as the cell wall component-lignin. Pretreatment with exogenous
JA may provide a mechanism of inducing ROS-scavenging prior to the damaging
effect of peroxidation, thereby protecting the photosynthetic mechanism and
membranes of the cell and moderating the damaging effects of drought.
This preliminary experiment was conducted under standard osmotic conditions with
a simplified objective: to determine the concentration of jasmonic acid that most
effectively instigates significant affects on biomass accumulation and PAL and
HMGR enzyme activity in switchgrass. Data from the experiment were utilized to
formulate an appropriate procedure for the application of jasmonic acid in
switchgrass. In addition, methods of enzyme extraction and analysis were evaluated
and refined in anticipation of future research in simulated drought conditions.
Materials and Methods
Experimental design: Growth and treatment of the experiment took place on the roof
of Howell Hall at the University of Central Oklahoma. The experimental design
consisted of four repetitions, each of which was arranged in a randomized block
design. On June 01 2010, the pots were seeded with 2.582 g switchgrass. The
switchgrass germinated on June 06 and the plants were thinned to 100 seedlings per
pot on June 12. On June 28 and July 18 a granular fertilizer was applied for a total
N treatment of 150kg/hectare, a total P treatment of 60 kg/hectare and a total K
treatment of 30 kg/hectare.
Dry weight (grams)
120.00
100.00
a
80.00
ab
ab
60.00
40.00
b
20.00
Figure 1: Treatment application
0.00
Harvest: On consecutive days between July 33 an July 33, the plants were
harvested. The plants were cut at pot level and weighed immediately to determine
fresh biomass. To obtain dry biomass, the plants were placed in a paper bag and
dried at 45° C for 2 days and weighed.
Introduction
Research involving the production of biofuel has been spearheaded in recent years
by the impending decline in fossil fuel availability and environmental concerns
associated with fossil fuel production and use. ‘Alamo’ and ‘Kanlow’ cultivars of
switchgrass (Panicum virgatum L.) are potentially efficient and economical crops
for the production of biofuel (Cassida et al. 2005). However, under drought
conditions, ‘Alamo’ shows a 49% decrease in biomass yield and ‘Kanlow’ shows
a 27% decrease in biomass yield (Stroup et al. 2003). Current research conducted
by our group is focused primarily on the optimization of bioenergy crop yield and
increasing the cultivar ’ s physiological tolerance to drought thereby enabling
expansion of the range of propagation to underutilized, semi-arid lands. To enable
economically feasible production a simple and effective method of combating the
negative effect of drought on these cultivars is necessary.
Biomass (DW)
140.00
0.0 mM
0.5 mM
1.5 mM
5.0 mM
JA treatment concentration
Figure 8. spectrophotometric procedure
Figure 5. JA effect on biomass accumulation
Values with the same letter are not significantly different
Results and Discussion
Analysis of the results indicated that the highest concentration of JA (5.0 mM)
significantly affected biomass accumulation (Figure 5). The 0.5 mM concentration
and the 1.5 mM concentration conveyed the general trend of decreased biomass with
the addition of JA application however, the results were not significant (Figure 5).
PAL
a
b
c
6
d
Figure 2. Switchgrass JA treatments replication 1 a. 0.0 mM b. 0.5 mM c. 1.5 mM
d. 5.0 mM
Enzyme extraction: The basal 10 cm of plant material was removed from each
sample and put on ice for enzyme extraction. The fresh samples were immediately
homogenized in a 50 mM Tris buffer (pH7.0) containing 0.1 M sucrose, 1%
polyvinylpyrrilodone, 4 mM cysteine and 1 mM DTT. The resulting products were
strained through 4 layers of cheese clothe and centrifuged at 5,000 g for 10 minutes
enabling isolation of the microsomal fraction containing HMGR, the supernatant
was removed and the pellet discarded. The supernatant was centrifuged at 10,000g
for 10 minutes enabling subcellular isolation of the cytosol containing PAL.
a
b
c
d
e
f
g
The JA treatment had a significant effect on PAL activity; the 0.5 mM concentration
and the 5.0 mM concentration elicited a significant decrease in PAL activity. The 1.5
mM concentration induced a reduction in activity however, the effect was not
significant. The JA treatment had a significant effect on HMGR activity; the 5.0 mM
concentration elicited a significant increase in activity while the 0.5 mM and 1.5 mM
concentrations induced significantly decreased activity compared to the control.
a
micromoles/gFW/hour
‘Alamo’ and ‘Kanlow’ switchgrass (Panicum virgatum L.) cultivars are highyielding perennials with substantial lignocellulosic content applicable for biofuel
production. However, these cultivars are indigenous to moist, lowland areas and
possess negligible tolerance to drought conditions. A physiochemical effect of
drought in plants is the generation of “reactive oxygen species” (ROS), which
damage membranes, proteins, and nucleic acids of the cell. The oxylipin hormone,
jasmonic acid (JA) may be exogenously applied as potentially effective treatment
to enhance drought tolerance through elicitation of ROS-scavenging enzyme
activity. An initial study was conducted to determine effective JA concentrations
for elicitation of significant changes in biomass yield and enzyme activity in
switchgrass. Four concentrations of JA (0.0 mM, 0.5 mM, 1.5 mM and 5.0 mM)
were evaluated by assessing biomass yield and the activity of enzymes essential for
production of phenolics (phenylalanine ammonia lyase; PAL) and terpenoids
(hydroxylmethyl glutaryl CoA reductase; HMGR). Results indicated a significant
effect on biomass accumulation in some JA treatments. Results also showed that
PAL activity decreased and HMG-CoA activity increased as a result of JA
application. Data from this experiment will be utilized to formulate a reliable
method of JA treatment applicable for increasing switchgrass tolerance to drought
conditions.
Treatment: On July 12 the plants were treated with Jasmonic acid. Neat Jasmonic
acid was purchased from Sigma-Aldrich, 50 mg of JA was dissolved in 5 ml of
methanol. From the concentrated solution, 0.0 mM, 0.5 mM, 1.5 mM and 5.0 mM
treatment solutions were prepared. Triton X was added to the solutions to act as a
superfactant enabling absorption of the compound into the intracellular space.
Treatment consisted of 10 mL dosages of treatment solutions applied via a spray
bottle to the leaves and stems of the plants. Treatment resulted in the application of
0.0 mmol, 0.005 mmol, 0.015 mmol or 0.05 mmol of JA per pot respectively.
5
ab
4
b
b
3
2
1
0
0.0 mM
h
0.5 mM
1.5 mM
JA treatment concentration
5.0 mM
Figure 6. JA effect on PAL activity
Values with the same letter are not significantly different
25
Figure 3. centrifugation products rep 1 a. switchgrass 0.0 mM microsomal fraction
b. Switchgrass 0.0 mM cytosolic fraction c. switchgrass 0.5 mM microsomal
fraction d. switchgrass 0.5 mM cytosolic fraction e. switchgrass 1.5 mM microsomal
fraction f. switchgrass 1.5 mM cytosolic fraction g. switchgrass 5.0 mM
microsomal fraction h. Switchgrass 5.0 mM cytosolic fraction.
Enzyme Assay: A spectrophotometric assay was conducted to determine the
activity of the PAL enzyme. Phenylalanine ammonia lyase enables efficient
production of trans-cinnamic acid from phenylalanine, this reaction is
spectrophotmetrically observed as an increase in the absorbance of light at 290 nm.
A spectrophotometric assay was conducted to determine the activity of the HMGR
enzyme. Hydroxymethyl glutaryl CoA reductase oxidizes NADPH and reduces 3hydrox-3-methylglutaryl-CoA to produce NADP+ and Mevalonic acid . This
reaction is spectrophotometrically observed as a decrease in the absorbance of light
at 280 nm indicative of a decrease in the concentration of the conjugated HMG-CoA
substrate and as a decrease in the absorbance of light at 340 nm indicative of the
oxidation of NADPH to NADP+.
a
20
b
c
Jasmonic acid is endogenously produced as a stress hormone, the application of
exogenous jasmonate induces a simulated, stressed condition resulting in decreased
primary productivity and decreased activity of PAL, an enzyme essential for lignin
production. However, high levels of JA increase the activity of enzymes for the
production of defensive compounds. The average endogenous concentration of JA
in a drought-stressed plant is ~0.5 mM, the exogenous application of 0.5 mM, 1.5
mM, and 5.0 mM evaluated the effect of JA at 1X, 3X and 10X the natural
concentration. Results suggest that application of high concentrations of jasmonic
acid diverts resource allocation from biomass accumulation to the production of
secondary protective compounds in a concentration dependent manner. Under
conditions of drought, application of 5.0 mM JA may increase biomass accumulation
over untreated samples due to the increased activity of HMGR for the production of
antioxidative terpenoids which serve to alleviate the deleterious effects of ROS
production in drought-stressed switchgrass. Further study in necessary to determine
whether high concentrations of JA effectively increases drought tolerance in
switchgrass.
Acknowledgements
HMG CoA
micromoles/gFW/hour
Abstract
Funding for this project was provided by a Research, Creative, and Scholarly Activities
(RCSA) grant from the Office of Research and Grants at the University of Central Oklahoma.
Seed for the project was generously donated by Johnston Seed Company.
bc
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10
5
0
0.0 mM
0.5 mM
1.5 mM
JA application concentration
Figure 7. JA effect on HMGR activity
Values with the same letter are not significantly different
5.0 mM
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