Transcript Effect of Jasmonic Acid on Biomass and Enzyme Activity in

Effect of Jasmonic Acid on Biomass and Enzyme Activity in Basil, Catnip, and Sage
Jocelyn Bidlack and Jim Bidlack
Department of Biology, University of Central Oklahoma, Edmond, OK 73034
Abstract
Four treatments of jasmonic acid (JA) (0.0 mM, 0.5 mM, 1.5 mM, and 5.0 mM)
were exogenously applied to basil (Ocimum basilicum), catnip (Nepeta
cataria), and sage (Salvia officinalis) to assess the effect of species and JA
treatment on biomass and activities of phenylalanine ammonia lyase and
hydroxymethyl-glutaryl CoA reductase. Results revealed that species had a
significant effect on FW, DW and percent moisture, suggesting that JA may
alter yield and moisture retention of species within the family Lamiaceae.
Results also indicated that JA did not have a significant effect on FW, DW, or
percent moisture in basil, but did affect measurements in other species. The 5.0
mM JA treatment resulted in a significant increase in percent moisture for
catnip. The 1.5 mM and 5.0 mM JA treatments revealed significant increases in
FW, DW and percent moisture for sage. Significant differences among
measurements, as affected by species and some species x treatment interactions
provides preliminary information regarding JA’s effect on growth dynamics in
the family Lamiaceae. Enzyme activities are currently being analyzed for
relative differences among species as well as the species x treatment
interactions.
Plants respond to environmental stress such as pathogens, herbivory, or
mechanical injury by the production of defensive secondary compounds such as
alkaloids, terpenoids, and phenolics. Desiccation of organelle membranes,
caused by mechanical or pathogenic wounding, results in the endogenous
production of jasmonic acid via the octadecanoid pathway (Creelman 1992).
Jasmonic acid is a naturally-occurring compound that acts as an elicitor in
metabolic pathways leading to the production of defensive secondary
compounds (Farmer and Ryan 1990). The exogenous 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). The effects vary
depending on the concentration of the jasmonic acid treatment; high
concentrations induce senescence and leaf abscission while lower
concentrations alter protein and mRNA composition (Mason and Mullet 1990).
This project was designed to assess how the exogenous application of four
concentrations of jasmonic acid affects biomass and enzyme activity in three
species: Ocimum basilicum, Nepeta cataria, and Salvia officinalis. The
objectives were to determine: 1) the effect of species, JA treatment, and
species x JA interactions on biomass accumulation, 2) the effect of species, JA
treatment, and species x JA interactions on enzyme activity, and 3) what
concentration(s) of JA significantly affect(s) biomass and enzyme activity
within species.
[JA]
0.0 mM
0.5 mM
1.5 mM
5.0 mM
Figure 5 Replication 1 Ocimum basilicum JA treatments shown in order from left
to right: 0.0 mM, 0.5 mM, 1.5 mM, and 5.0 mM.
•Enzyme extraction: The top three internodes and bottom three internodes of
each sample were isolated for enzyme extraction. The fresh 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
6) to enable subcellular isolation of: 1) microsomes containing hydroxymethylglutaryl CoA reductase and 2) cytosol containing phenylalanine ammonia lyase.
Figure 2. Replications 1 and 2 at eight weeks.
Introduction
Table 3. Biomass and moisture of species as affected by JA.
•Experimental design: The growth and treatment of the experiment took place
on the rooftop greenhouse of Howell Hall at the University of Central
Oklahoma. The experimental design consisted of four replications, in each
replication three species and four treatments were arranged in a randomized
block design (Figure 2). Borders consisting of untreated basil, catnip and sage
were used to minimize environmental variation. On 28 March, 18 June, and 3
July 2010, the plants were fertilized with miracle grow 15:30:15 liquid
fertilizer for a total N treatment of 162 kg/hectare, a total P2O5 treatment of 325
kg/hectare and a total K2O treatment of 162 kg/hectare.
•Cutting for Regrowth: To account for variation in germination time for these
species, plants were grown for eight weeks following transplant and then cut
back to an approximate height of 3.0 centimeters on 10 June 2010. Subsequent
regrowth was utilized in this experiment to ensure that the age of the material
analyzed was standardized.
•Treatment: Jasmonic acid (JA) was applied to the samples twice; the first
application occurred approximately four weeks after the cutback date on 5 July
and the second application occurred approximately three weeks before harvest
on 19 July 2010. “Neat” JA was purchased from Sigma Aldrich and 50 mg of
JA was dissolved in 5.0 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 surfactant enabling absorption of the
compound through the cellular membrane. Treatment consisted of two 5.0 mL
dosages of treatment solutions applied via a spray bottle to the leaves and stems
of the plants (Figures 3, 4, and 5). Treatment resulted in the total application of
0.0 mmol, 0.005 mmol, 0.015 mmol or 0.050 mmol of JA per pot, respectively.
•Harvest: On consecutive days between August 9 an August 12, replications of
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. Percent
moisture was calculated from these results.
a
b
c
d
e
f
g
Figure 6. Replication 1 Ocimum basilicum centrifugation: a. top internodes 0.0
mM, b. basal internodes 0.0 mM, c. top internodes 0.5 mM,
d. basal internodes 0.5 mM, e. top internodes 1.5 mM, f. basal internodes 1.5
mM, g. top internodes 5.0 mM, and h. basal internodes 5.0 mM.
•Enzyme Assay: A spectrophotometric assay 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. Hydroxymethylglutaryl 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.
.
Table 1. Analysis of variance for biomass and moisture.
a
b
c
d
Figure 3. Replication 1 Nepeta cataria JA treatments shown in order from left
to right: 0.0 mM, 0.5 mM, 1.5 mM, and 5.0 mM.
Sample
Species
Replication
Error A
JA
JA x Species
FW
**
NS
**
NS
NS
DW
**
**
**
NS
NS
% moisture
**
NS
**
**
**
**Significant at the p < 0.05 level; NS = not significant.
Table 2 . Biomass and percent moisture of species.
a
b
c
d
Figure 1. Replication 1 following transplant.
Figure 4. Replication 1 Salvia officinalis JA treatment shown in order from left
to right: 0.0 mM, 0.5 mM, 1.5 mM, and 5.0 mM.
Species
Basil
Catnip
Sage
FW (g)
98.75 a†
58.13 b
26.63 c
DW (g)
20.44 a
16.44 b
10.50 c
0.0 m M
0.5 mM
1.5 mM
5.0 mM
DW (g)
% moisture
------------------------------- Basil -----------------------------107.5 a†
22.00 a
78.95 a
100.0 a
20.00 a
79.94 a
82.50 a
18.25 a
74.44 a
105.0 a
21.50 a
79.38 a
------------------------------- Catnip ---------------------------58.75 a
18.00 a
69.18 b
55.00 a
16.25 a
70.33 b
57.50 a
16.00 a
72.29 ab
61.25 a
15.50 a
74.48 a
------------------------------- Sage -----------------------------18.00 b
8.750 c
48.08 b
22.00 b
10.00 bc
53.46 ab
32.75 a
11.25 ab
63.36 a
33.75 a
12.00 a
63.33 a
†Means for each species within a column followed by the same letter are not
significantly different.
Results and Discussion
h
Materials and Methods
• Planting and Maintenance: Three species of family Lamiaceae were chosen
for this project. Seeds were purchased locally (TLC Greenhouse & Nursery)
and planted in flat trays with sterilized potting soil. Germination took place in
an environmental chamber located in Howell Hall. After three weeks, the
plants were transplanted to pots with a diameter of 15.2 cm and moved to a
greenhouse for the duration of the experiment (Figure 1). Three weeks after
transplanting, the plants were thinned to three plants per pot.
0.0 mM
0.5 mM
1.5 mM
5.0 mM
FW (g)
% moisture
78.18 a
71.57 b
57.06 c
†Means followed by the same letter within a column are
not significantly different.
Analysis of variance revealed significant differences in fresh weight (FW), dry
weight (DW), and percent moisture as affected by species as well as percent moisture
for the species x jasmonic acid (JA) interaction (Table 1). Further analysis using least
significant difference (LSD) tests showed that basil demonstrated significantly higher
fresh and dry weight compared to both the catnip and sage (Table 2). Catnip
demonstrated significantly lower FW, DW, and percent moisture compared to basil
but significantly higher values for these measurements compared to sage (Table 2).
Sage demonstrated significantly lower FW, DW, and percent moisture compared to
both basil and catnip (Table 2). Differences in growth habits among the species likely
account for the significant differences in FW, DW and percent moisture observed.
Jasmonic acid significantly affected percent moisture in some species according to
ANOVA (Table 1). Significant differences in percent moisture, as affected by JA,
suggested that JA has potential to increased water retention, and hence, may be used
to promote drought resistance in these species. Further analysis using LSD suggests
that JA may have species-specific effects on FW, DW and percent moisture (Table 3).
For instance, JA did not significantly affect FW, DW or percent moisture of the basil,
but JA did significantly affect percent moisture for catnip and also significantly
affected all measurements in sage (Table 3).
In catnip, the 5.0 mM JA produced significantly higher percent moisture compared
to the 0.0 mM and 0.5 mM JA treatments in the same species (Table 3).
In sage, the 5.0 mM and 1.5 mM JA treatments produced significantly higher FW
compared to 0.5 mM and 0.0 mM JA treatments and the 5.0 mM JA produced
significantly higher DW compared to the 0.5 mM and 0.0 mM JA treatments in the
same species. Interestingly, the higher concentrations of JA used for sage produced
increased biomass even though JA did not affect biomass in the other species.
The effect of high JA treatments on biomass is a valuable observation providing:
1) information on the mechanism of the JA action, and 2) guidance concerning
treatment concentrations in further studies. The effect of JA treatment on moisture
retention and biomass are promising indications of disease and pest resistance as
affected by JA treatment. The enzyme activities of phenylalanine ammonia lyase and
hydroxymethyl-glutaryl CoA reductase are currently being analyzed to evaluate
differences among species as well as the species x JA interaction.
Acknowledgements
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.
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