Effect of Jasmonic Acid on Biomass and Enzyme Activity in Switchgrass and Sorghum
Jocelyn Bidlack and Jim Bidlack
Department of Biology, University of Central Oklahoma, Edmond, OK 73034
Abstract
Optimization of biomass yield in switchgrass (Panicum virgatum) and sorghum
(Sorghum bicolor) is essential for efficient and economical production of biofuel.
Four treatments of jasmonic acid (JA) (0.0 mM, 0.5 mM, 1.5 mM, and 5.0 mM)
were applied to assess the effect of species, treatment, and the species x
treatment interaction on biomass and the activities of enzymes involved in
development of factors affecting pest resistance. Results indicated that fresh
weight (FW), dry weight (DW) and percent moisture varied significantly among
species. The 5.0 mM concentration of JA resulted in decreased FW and DW in
switchgrass, and decreased FW in sorghum. Significant differences, as affected
by species and the species x treatment interaction, suggest that species selection
and JA treatment are worthwhile considerations when evaluating biomass yield
of these species. Interestingly, the high JA treatment decreased production in
both species, which may be an important factor concerning treatment
concentrations in further studies. The lack of significant effects on biomass in the
lower JA treatments provides indication of potential use of this growth regulator
for pest resistance without decreased biomass. Additional experiments are being
investigated on activities of phenylalanine ammonia lyase and hydroxymethylglutaryl CoA reductase to determine how JA may or may not be affecting
enzymes affecting pest resistance.
•Experimental design: The growth and treatment of the samples took place on the
roof of Howell Hall at the University of Central Oklahoma. The experimental
design consisted of four replications, each consisting of two species and four
treatments arranged in a randomized block design (Figure 2). Borders, composed of
untreated switchgrass and sorghum, were used to reduce the effect of differing
environments on the plant growth. On June 01 2010, the pots were seeded with
2.582 g (3000 seeds) switchgrass or 0.668 g (25 seeds) of sorghum. The sorghum
germinated on June 03, at which time ~10-15 seedlings emerged. The switchgrass
germinated on June 06, at which time ~300 seedlings emerged. The seedlings were
thinned on June 12; the switchgrass was thinned to 100 seedlings per pot and the
sorghum was thinned to 10 plants per pot. On June 18th, the plants were fertilized
with miracle grow 20:20:20 liquid fertilizer, and on June 28 and July 18 a granular
29-0-5 fertilizer was applied for a total N treatment of 238 kg/hectare, a total P2O5
treatment of 73 kg/hectare and a total K2O treatment of 113 kg/hectare.
As an alternative to these traditional remedies, the application of jasmonic acid
has been implemented as an environmentally safe and effective method of
endowing pest resistance to many commercial crops including corn, potato, and
tomato (Cohen et al. 1993, Schmelz et al. 2003, Thaler 1999). Jasmonic acid is
a naturally-occurring compound that acts as an elicitor in metabolic pathways
leading to the production of defensive secondary compounds. The exogenous
application of low concentrations of jasmonic acid effectively induces the
production of these desirable defensive compounds (Mason and Mullet 1990);
in the event of infestation, treatment may increase the yield of these valuable
alternative crops. Further research is needed to determine the effect of
exogenous JA on switchgrass and sorghum.
The objectives of this experiment were to determine 1) the effect of species and
jasmonic acid treatment on the biomass of switchgrass and sorghum, 2) the
effect of species and jasmonic acid treatment on the activity of enzymes
involved in the production of defensive secondary compounds and 3) what
concentration(s) of jasmonic acid treatments significantly affect biomass within
species.
Figure 2. Examining the experimental design.
•Treatment: On July 12 the plants were treated with jasmonic acid (JA) (Figure 4).
Neat JA 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-100 was added to the solutions to act as
a surfactant enabling absorption of the compound through the cellular membrane.
Treatment consisted of 10 mL dosages of JA 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.
b
c
d
e
f
g h
•Enzyme Assay: A spectrophotometric assay (Figure 6) was conducted to
determine activity of the PAL enzyme. Phenylalanine ammonia lyase catalyzes
production of trans-cinnamic acid from phenylalanine; this reaction is
spectrophotmetrically observed as an increase in the absorbance of light at 290
nm. Hydroxymethyl-glutaryl CoA reductase catalyzes oxidation of NADPH and
reduction of hydroxymethyl-glutaryl CoA; this reaction is spectrophotometrically
observed as a decrease in the absorbance of light at 340 nm.
.
Figure 6. Spectrophotometric procedure.
Figure 3. Harvest of replications 1 and 2.
Table 1. Analysis of variance for biomass and moisture.
On 17 May 2010, switchgrass seeds were obtained from the USDA-ARS
Grazinglands Research Laboratory in El Reno, Oklahoma. Sorghum seeds
were purchased from Ross Seed Company in El Reno on the same day.
a
b
c
d
Sample
Species
Replication
Error A
JA
JA x Species
FW
**
**
NS
**
NS
DW
**
**
NS
NS
NS
% moisture
**
NS
NS
NS
NS
**Significant at p < 0.05 level; NS = not significant.
Table 2. Biomass and percent moisture of species.
e
Figure 1. Germination study.
f
g
h
Figure 4. JA treatments for replication 1 . Switchgrass: a. 0.0 mM, b. 0.5 mM, c. 1.5
mM, and d. 5.0 mM. Sorghum: e. 0.0 mM, f. 0.5 mM, g. 1.5 mM , and
h. 5.0 mM
Table 3. Biomass and moisture as affected by JA.
[JA]
0.0 mM
0.5 mM
1.5 mM
5.0 mM
0.0 mM
0.5 mM
1.5 mM
5.0 mM
•Harvest: On consecutive days between July 33 an July 33, the plants were
harvested (Figure 3). 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 then weighed. Percent moisture was
calculated from these results.
Materials and Methods
•Germination study: To determine the germination rates of the two species, a
preliminary germination study was conducted (Figure 1). Two Petri dishes were
lined with moistened filter paper and 10 seeds of a given species were deposited
on the dish and covered for three days. Four repetitions of the study were
completed and the average germination rate for each species recorded for use in
the seeding rate calculation. The average germination rates for switchgrass and
sorghum were 10% and 80%, respectively.
a
Figure 5. Centrifugation products from Replication 1: a. switchgrass 0.0 mM,
b. sorghum 0.0 mM, c. switchgrass 0.5 mM, d. sorghum 0.5 mM, e. switchgrass
1.5 mM, f. sorghum 1.5 mM , g. switchgrass 5.0 mM, and h. sorghum 5.0 mM.
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. The benefits of
switchgrass (Panicum virgatum L.) and sorghum (Sorghum bicolor (L.)
Moench) as potentially efficient and economical crops for the production of
biofuel have been expounded in recent studies (Antonopoulou et al. 2008,
Cassida et al. 2005). Optimization of these crops’ yield is essential for the
eventual transition of biofuel production from experimental to commercial
applications. Crop yield is significantly hindered by disease and pest
infestation; however the spraying of fungicides and pesticides is expensive,
requiring multiple treatments significantly contributing to the cost of biofuel
production. In addition, fungicides and pesticides are corrosive chemical
compounds that contribute to the degradation of the environment.
•Enzyme extraction: The basal 10 cm of plant material was removed from each
sample and stored on ice for enzyme extraction. The samples were immediately
homogenized in a 50 mM Tris buffer (pH 7.0) containing 0.1 M sucrose, 1%
polyvinylpyrrilodone, 4 mM cysteine and 1 mM DTT. The resulting products
were strained through 4 layers of cheese cloth and centrifuged (Figure 5) to
obtain subcellular isolation of: 1) the microsomes containing hydroxymethylglutaryl CoA reductase and 2) cytosol containing the phenylalanine ammonia
lyase .
Species
Switchgrass
Sorghum
FW (g)
296.3 b†
1454 a
DW (g)
62.50 b
419.2 a
% moisture
79.67 a
71.11 b
†Means followed by the same letter within a column are
not significantly different.
FW
DW
% moisture
--------------- Switchgrass ---------------391.7 a†
90.00 a
81.97 a
355.0 a
70.00 ab 79.87 a
270.0 ab
56.67 ab 79.27 a
168.3 b
33.33 b
77.56 a
---------------- Sorghum ------------------1516 a
445.0 a
73.19 a
1508 a
416.7 a
72.51 a
1472 a
415.0 a
69.67 a
1322 b
400.0 a
68.89 a
†Means for each species within a column with the same
letter are not significantly different.
Results and Discussion
Analysis of variance revealed significant differences in fresh weight (FW),
dry weight (DW), and percent moisture as affected by species, and differences
in FW as affected by the species x JA interaction (Table 1). The FW and DW
of sorghum were significantly higher than FW and DW of switchgrass (Table
2). Differences in biomass were expected between species because of
differences in annual and perennial growth habits. Switchgrass, a perennial, is
unlikely to devote energy to extensive biomass accumulation for a single year,
whereas sorghum, an annual, relies on biomass accumulation for competitive
reproduction and survival within a single year.
Replication had a significant effect on the FW and DW of the samples
(Table 1). These differences were probably due to varying effects of shading
by sorghum on bordering and medial replications. Replication 4 was
discarded from analysis because of discrepancies in fertilization rates and
differing environmental influences throughout the experiment.
The JA treatment had a significant effect on fresh weight (FW) in both
species according to the ANOVA (Table 1). Further analysis using LSD
suggested that in switchgrass, the 5.0 mM treatment of JA resulted in
significantly lower FW compared to the control and 0.5 mM treatments, and
significantly lower DW compared to the control treatment (Table 3). The LSD
analysis also suggested that in sorghum, the 5.0 mM JA treatment resulted in
significantly lower FW compared to the control, 0.5 mM and 1.5 mM
treatments (Table 3). The inhibitory effect of the high JA treatment is a
valuable observation providing guidance concerning treatment concentrations
in future studies. The 5.0 mM treatment consisted of the application of 5
mmol of JA to each pot regardless of growth habit, thus the moles per unit cm
of plant material was much higher for the switchgrass compared to sorghum,
causing the effect of the treatment to be exaggerated in the switchgrass .
The enzyme activities of phenylalanine ammonia lyase and hydroxymethylglutarayl CoA reductase are currently being analyzed for relative differences
in species and species x JA treatment.
The experiment yielded important preliminary data for future investigation
of JA’s effect on yield and pest resistance in these valuable crops.
Acknowledgements
We thank Charles MacKown of the USDA-ARS Grazinglands Research
Laboratory for providing switchgrass seeds and for advice throughout the
experiment. Funding was provided by the Experimental Program to Stimulate
Competitive Research (EPSCoR) – Research Experience for Undergraduates
(REU) program.
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