Download Acid Rain Effects on the Production and Composition of Epicuticular

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Acid Rain Effects on the Production and Composition of
Epicuticular Waxes of Turf Grass, Poa-pratensis-lolium-festuca
Natalie Buch
Syracuse University, Syracuse NY
Mentors: Maureen Conte and JC Weber
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Abstract
An experiment was performed for 1 week to determine the physiological and morphological
response of Poa-pratensis–lolium-festuca to simulated acid rain. Five pH treatments
(5.6,4.4,3.5,2.4 and 1.6) were selected to examine if any differences in wax production and
composition resulted from this stress. The five treatments were chosen to resemble real pH
rainwater values -unpolluted rainwater pH or control, the current northeastern US pH, the current
Hong Kong pH, the lowest pH recorded in Europe and the lowest recorded in the US,
respectively. The waxes were quantified and identified with a GM/MS after being oven dried,
ultrasonicated with an organic solvent, centrifuged, folch’s extracted, transesterified, and finally
derivatized the samples. There was a slight discrepancy between the control and the rest of the
treatments in the total new growth, however no trend in growth or biomass with increasing
acidity. pH 1.6 had a lower C:N molar ratio, this treatment also had a higher total wax
concentration –confirmed with the SEM qualitative analysis- and an increase in the longer chain
distributions of alcohols and acids. The vegetation was characterized by high abundance of C 26 nalcohol. The results of relative distribution of class and chain length of compounds were very
similar and reproducible, but the results suggest there was subtle change as a physiological
response. No trends of the isotopic fractionation appeared with increasing acidity, indicating the
stomatal mechanism was not altered.
Keywords
Acid rain, leaf waxes, wax class composition, chain length distribution
Introduction
As anthropogenic pollution, mainly from fossil-fuel combustion, continues to be released
into the atmosphere acid deposition comes along as a product of Sulfur dioxide and Nitric oxides
after being oxidized in the atmosphere. This term is applied to precipitation with a pH below 5. In
the United States and more specifically the northeast, acidification values have decreased since
the Clean Air Act (1990), however the global emissions have increased. Asia, Africa, Canada and
South America are more likely to continue increasing the discharge given the reality of
population growth and their potential for industrial expansion (Galloway, 1989). High levels of
sulfuric, but also nitric acid can affect the growth of vegetation, leaf and species composition, soil
buffering capacity among other components of terrestrial ecosystems (Larssen et al, 2006).
Plants protect their aerial surfaces with an external hydrophobic cuticle layer, coated with
epicuticular wax, which acts as an obstacle to drought, low temperature, UV-B radiation and
pathogens and herbivores (Maffei, 1996). Since waxes are at the interphase with the atmosphere,
they also play an important role in gas exchange by reducing transpiration.
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The morphology of the waxes is mainly influenced by growth conditions (Baker and Hunt,
1986). The crystalline deposits that overlay the cuticle vary in forms, such as plates, ribbons,
tubes and rods (Baker, 1982). Variations in wax morphology may be caused by differences in the
chemical composition of the material or different arrangement of the same compounds. The
composition of the leaf wax is subject to continuous change as leaves expand and constantly
ablate by natural exfoliation, wind and dust abrasion and regenerate the waxes (Hadley and Smith
1989; Rogge et al. 1993). They also vary with species, genus and parts within a plant according to
their ecological function in the environment (Eigenbrode, 1995).
These waxes are formed when the epidermal cells secrete fatty acids that polymerize from
exposure to oxygen. They are a complex heterogeneous mixture of long-chain (C20-C34) fatty
acids and their derivatives that during biosynthesis are further modified into major aliphatic
components (Rhee et al, 1998), mainly hydrocarbons, esters, primary alcohols, triacylglycerols,
fatty acids, ketones and aldehydes, (Barthlott et al, 1998) that reflect the episodic nature of the
transported atmospheric material.
Manipulation of the acidity of the water source could be used as a projected response of
plants to the possible future advection of weathering (Baker and Hunt, 1986) and pollutants, for
waxes are indicators of air pollution effects (Turunen, et al 1997). Growth will also be monitored
to estimate biomass differences among treatments. The SEM will allow me to compare and
understand the morphological changes of the wax structure and distribution on the leaf surface.
Imaging the conglomerations that form in the cells that surround the stomata can tell more about
wax production and crystals of the grass. A qualitative examination of the stomatal openings
should reflect changes in stomatal conductance and therefore determine carbon fractionation,
even though terrestrial photosynthesis discriminates against 13CO2 (Conte and Weber, 2002), if
plants are stressed they tend to utilize less 12CO2 and have a heavier carbon signal (Igamberdiev,
2003).
Waxes are not only important in an individual level because they define the surface
properties of the leaf, but can also affect the secondary production of its ecosystem. The effects of
their composition can affect grazers and upper trophic levels that obtain energy from this plant
source. An example of this is how cuticle thickness interferes with bacterial colonization of the
plants (Lindow et al, 2003). A lower relative abundance of waxes can lead to modifications due
to more microbial activity. The extent at which the cuticle is being affected should be assessed
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because leaves become less hydrophobic following exposure to gaseous air pollutants and
become more susceptible to nutrient leaching (Percy and Baker, 1987). Air pollutants also alter
the structure and arrangement of the waxes, and this can result in wax erosion (Riding and Percy,
1985).
Waxes are biologically important, but their role protecting plants is of economic interest
because they have a commercial value (Seigler, 1998), they are important for the pharmaceutical,
cosmetic and food industries (Olubunmi, 2010). Understanding the changes in wax production
and composition also has major implications and use in paleo studies. Poaceae are a source of
biomass in the geological record of soils, as well as lake and marine sediments (Rommerskirchen,
2006). Grasses are useful for this experiment, for they have a rapid growth rate. The main wax
component of grasses are alcohols or esters, in rainforests long chain alkanes are more abundant
(Tulloch and Hoffman, 1977; Vogts et al, 2009). In addition they are a major food source for both
humans, grazing and domestic animals.
Objectives
My objectives include studying how different pH levels of wet acid deposition affect
epicuticular waxes of leaves by measuring total wax biomass, wax concentrations, component
class composition of n-alkanes, n-alcohols and n-acids, and carbon and nitrogen abundance and
isotopic fractionation. The leaf phyllosphere will also be examined to correlate it to the
physiological results.
Materials and methods
Sod grass (Poa pratensis- lolium- festuca) was purchased from Mahoney’s Garden Center
in East Falmouth MA, put into pots, and allowed to acclimate in a growth chamber for one week
before the experimental simulated precipitation treatment began. At the start of the experimental
phase, the grass was cut to the lowest extent possible to be able to measure the new growth. The
grass was under 200 C, and received approximately 1,550 µE m−2 s−1 (medium-high setting in
the chamber) for a 16-h light period per day. As for simulated precipitation, five treatments with
pH values of 5.5, 4.4, 3.5, 2.5, and 1.5 were prepared by dilution of reagent grade sulfuric acid
and nitric acid (70% and 30%, respectively) with deionized water. Rainfall was simulated
applying the acid solutions from hand-held atomizers once a day between 20:30 and 21:00. I
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selected this period of time because the light of the growth chamber turned off at 21:00; this way
I was reducing evapotranspiration. 75ml of water was calculated to be 2x the average rainfall (0.1
inches) per area (6.5 cm) in the northeast, and it was supplemented with spritzers that contained
the acid treatments. These treatments were chosen for, pH 5.6 is the natural pH of unpolluted
rainwater, pH 4.4 is the average pH of northeastern US, pH 3.5 is the average rainfall pH in
Tseung Kwan O, Hong Kong and 2.4 is the lowest pH recorded in Europe (Scotland), and pH 1.5
is the lowest recorded in the US (West Virginia) . Soil pH was also measured, the first and the
final day of the experiment. The soil was collected and diluted with water at a 5:1 ratio and
measured by a Hannah pH probe.
After 7 days of growth, the grass was clipped and weighed to obtain the total wet biomass
of each treatment. I isolated 3 blades from each treatment for the SEM analysis before drying the
samples to obtain the dry mass. The isotopic and epicuticular composition of the grass species
was then to be examined. A total of 10 (three duplicates, two single samples, and a procedural
blank) samples were tested for extraction of waxes. Subsequently, they were all oven-dried at 5560°C overnight. The blades were grounded into a powder using a MeOH- rinsed mortar-pestle
and 50 mg of each sample were transferred into solvent rinsed 16 mm Pyrex tubes for
epicuticular wax extraction. At this phase, a subsample was taken (≈2 mg) for carbon and
nitrogen bulk abundance and stable isotope analysis using a Europa ANCA-SL elemental
analyzer preparation unit interfaced with a Europa 20-20 Continous-Flow Isotope Ratio Mass
Spectrometer in the Ecosystem Center’s Stable Isotope Laboratory.
An internal standard mixture containing compounds from each wax component class was
added to all the pyrex tubes, to be able to quantify the wax compounds. The concentrations of the
standard were 10.20 µg of 21 fatty alcohol, 10.28 µg of 5 alpha cholestane, 11.26 µg of 23 fatty
acid and 11.31 µg of 36 alkane. The ultrasonic extraction consists in immersing the dry material
in an organic solvent, dichloromethane (DCM) and ultra-sonicating twice with a Misonix
Ultrasonic Processor for ten minutes to extract the lipids. All samples were then centrifuged
around 3000 rpm and the DCM organic extract vacummed filtered through fritted funnels into
separatory funnels to eliminate transfer of leaf particulates. A Folch’s extraction was done using,
10 ml of 0.88% aqueous KCl to separate water-soluble proteins and carbohydrates from the
organic extract. The extracts were then passed though anhydrous sodium sulfate columns to
remove excess water that would interfere in the reactions of transesterification, when the complex
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molecules are broken down to component classes and free fatty acids are derivatized into methyl
esters.
The transesterification process occurs after resuspending the extracts in 0.5 ml toluene and
adding 2 ml transesterification reagent (10% methanolic HCL),(made with 30 ml of anhydrous
methanol, 3.0 ml of acetyl chloride). The result will be the phase separation of the glycerol from
the extract products. Esterification occurs after the reaction mixture is capped under nitrogen and
heated at 50°C overnight. After this step the fatty acids will convert to fatty acid methyl esters.
To remove the non-lipid contaminants from the extracts a hexane extraction was done, it consists
in shaking the mixture with an aqueous 5% NaCl solution (Christie, 1993) and 2ml of hexane.
The transesterified extracts were hexane extracted and evaporated to just dry using a Svant
SpeedVac and resuspended in DCM.
The next step will render highly polar materials to be sufficiently volatile without thermal
decomposition or molecular re-arrangement. During the derivatization process, the dry samples
were resuspended in 50µl pyridine and 50µl of the catalyst 1% TMCS/ trimethylchlorosilane and
the silylating reagent BSTFA/ N,O-bis(trimethylsilyl) trifluoroacetamide were added , capped
under nitrogen and heated at 55ºC for 1 hr. Derivatives react with active hydrogen atoms of the
sample and a trimethyl group is attached to the hydroxyl groups. Derivatization is required, for
fatty acids and fatty alcohols are not volatile, otherwise the GC could not monitor these (Orata,
2012). By derivatizing, the adsorption of the analyte in the GC is reduced and it will increase
detectability.
In a gas chromatography (a GC Agilent technologies 7890A model with a CPSil SCB
60m x 0.25mm x 0.25µm film column and a temperature program of 50(2) 150 320 (30) (for
a total of 85 min) volatile organic compounds are separated as a result of equilibria between the
solutes and GC column identifies and quantifies individual classes of waxes and molecular
species. Afterwards, a Mass Spectrometer (MS Agilent technologies 5975C with triple-Axis
detector) analyzed the components of the samples by charging the specimen molecules,
accelerating them through a magnetic field, breaking the molecules into charged fragments and
detecting the different charges (Christie, 2003). Chromperfect software (Justice Laboratories) was
later used to retrieve the peak areas of the FID readout that were used for quantification.
Before the Scanning Electron Microscopy (Zeiss Supra40VP) could be utilized to
examine the samples, a blade tip from the pH 1.6, 35 and 5.6 treatments were cut in small pieces
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and affixed to aluminum stubs by double-sided adhesive tape and later air-dried (Barthlott, 1998).
The stubs were then coated with a platinum layer. Both sides of a blade of the three treatments
will be observed, for a total of 6 samples.
Results The growth of Poa pratensis-lolium-festuca was slightly affected by the acid treatments,
the control had about 24% taller than the average of the rest of the treatments. There was not a
declining trend in the total growth (Fig.1). On average, the difference in biomass between the
least and most acidic treatment was 0.04 g per dry weight (Fig. 2). However, there was a notable
morphological difference between the pots of the pH 1.6 treatment and the rest; foliar damage
was evident in all the blades of this treatment.
As to elemental analysis, the percent C by dry weight decreased with increasing acidity
(Fig. 3). Nitrogen content increased with increasing acidity (Fig.4). For this, the lowest pH
treatment had a much lower carbon to nitrogen molar ratio and the ratios increased
logarithmically with increasing pH (Fig.5). As to isotopic fractionation, the variations in the δ 13
C and δ 15 N values did not correlate to expected results (Fig. 6, 7). The pH 3.5 and pH 5.6
treatments δ 15 N had high variability between replicates (>0.5 mils).
In all treatments n-alcohols were the dominant class component, the relative distribution
percentage of n-alcohols in the treatments ranged from 62 to 72% (Fig. 8). n-acids formed ~20%
of the waxes, whereas n-alkanes conformed ~10%. Treatment pH 1.6 had higher total wax
concentration to organic carbon ratio (Fig. 9). It had an average of 111.11 µg/g C; the treatments
pH 2.4, 3.5, 4.4 and 5.6 had 80.25, 86.49, 83.54, 80.73 µg/g C respectively after adding the FAL
24-32, FAME 20-34 and ALK 29-33.
The molecular distribution of longer chain alcohol and acid components increased with
acidity. 26 fatty alcohol in the wax on the leaves in the pH 1.6 treatment is ~21 µg/g C or 30%
higher (Fig. 10a) than the average of the rest of the treatments (Fig. 10b-10e). The long chained
C28 and C30 fatty alcohols had higher concentrations in the most acidic treatment (Fig. 11a).
Although there is variation between replicates, this treatment also had higher concentrations in
the longer fatty acid chain lengths, (Fig. 12) A higher abundance of longer chain compounds in
the C30-C34 n-acids correlates positively with the longer chain abundance of C30-C32 n-alcohols
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(Fig. 13). A peculiar outcome of the 1.6 pH treatment is seen in the alkane distribution of C29 and
C31 (Fig. 14). This treatment’s average resulted in a higher C29 in relation to C31 pH (Fig. 15), I
should note that there was greater variability in the concentration of alkanes in the lowest acidity
treatment. Another interesting result is the higher concentration of sterols in the pH 1.6 treatment
(Fig. 16).
The images retrieved from the SEM support the quantitative results of total wax; a more
uneven distribution was observed in the control treatment (Fig. 17b vs Fig. 19b). Less wax
conglomerates were seen near the midrib of the leafs (Fig. 17c ). No differences were noted in the
stomata among treatments (Fig. 17e vs Fig. 19c ). Crystalline structures were only present in the
most acidic treatment (Fig. 18e,f), these ranged between ~10 and ~30 µm. As to the intermediate
treatment, the wax distribution looked similar to the 5.6 pH treatment (Fig. 18b).
Discussion
It has been proven that plant species vary in their susceptibility to the effects of acid rain,
and Poa- pratensis-lolium-festuca had a physiological response after the period of one week. The
minor differences in growth and biomass are not surprising because this outcome has been
observed in many other studies, for growth is highly dependent on the soil’s buffering capacity
(Amthor, 1984). And, the soil utilized for the experiment was very alkaline (Table 1).
The trend of lower carbon percentage in relation to dry weight with increasing acidity has
been observed in an earlier study. Ferenbaugh’s data of Phaseolus plants in 1976, indicated that
the acidified plants sustained a loss of capacity to produce carbohydrates. A theory this author
gives is that this might due to a slight increase in the respiration rate that would not allow the
plant to catabolize the additional carbohydrate. For the pH 1.6, the decrease in carbon shows that
even though there was a higher wax concentration the acid rain is still affecting carbon abundance.
An explanation for the higher nitrogen abundance of the most acidic treatment could be explained
by the plant incorporating N from the nitric acid irrigation. However, a study done in the
Howland forest in Maine states that by incorporating N plants also increase carbon sequestration
and this was not the case for the C of my experiment. Nonetheless, a possibility is that since N
and dark respiration rates are related, they increase linearly (Osaki, 2001).
The relative distributions of the class components are very similar and reproducible, this
was expected, for the reason that they are characteristic of their genus and the molecular
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composition determines the three dimensional structure of the wax which can vary greatly among
different types of plants. The SEM images support the results of total wax concentrations, higher
wax concentrations in the most acidic treatment as a defensive response. Looking into the
molecular distribution of alcohols and acids, the trend of higher concentration in the longer chain
compounds suggests that the plant is subtly responding by producing longer and therefore, more
resistant and refractory compounds in order to protect itself from degradation. This effect has also
been observed after plants have been under water stressed conditions (Bondada, 1996). The
increase of total wax concentration of treatment pH 1.6 is also noteworthy; it’s another defensive
response after such a short period of time.
Yet, after qualitatively examining the stomata of treatments pH 1.6, 3.5 and 5.6 there were
no notable differences in the morphology. Which leads to the isotopic response; we would have
expected a higher fractionation in treatment pH 1.6. Perhaps, this indicates that the wax protected
the stomata enough that it did not affect the gas exchange process. I would speculate that if the
treatments were applied for a longer period of time, there would be a change in both the openings
and the 13CO2 signal. Perchance in a future study a more quantitative analysis could allow to
make more accurate comparisons between the acid treatments. Another factor that might interfere
with the material observed in the SEM is the lag time between the harvest time and the time when
the plants were affixed to the stubs. The technique to fix the samples might have also altered the
observed results.
It would be interesting to determine the implications this has for paleoenvironmental
reconstruction studies. Also, to examine if there is an equivalent response after being acidified by
dry deposition. Longer chain distributions could tell whether plants were under stressful
atmospheric conditions. Looking at class components shouldn’t be enough to determine
differences, for the plant will preserve its signature associated with its genus. But, the molecular
composition should also be studied. In conclusion, subtle changes in chain length can infer
changes in the precipitation’s water quality.
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Total growth blade growth (cm) 7 Figure 1.
6 5 4 3 2 1 0 0 Figure 1.
1 2 3 pH treatment 4 5 6 11
Total Biomass new biomass (g/dw) 0.45 0.4 0.35 Figure 2.
0.3 0.25 0.2 0.15 0.1 0.05 0 0 Figure 2.
1 2 3 pH treatment 4 5 6 12
% Carbon by dry weight 46 45 45 % C 44 44 43 43 42 42 41 41 40 0 1 2 3 4 pH treatment Figure 3.
5 6 13
% Nitrogen by dry weight 6.0 % N 5.5 5.0 4.5 4.0 3.5 0 Figure 4.
1 2 3 pH treatment 4 5 6 14
14 y = 2.6463ln(x) + 8.2179 R² = 0.91388 Mole C:N ratio 13 12 11 10 9 8 0 1 2 3 4 pH treatment Figure 5.
5 6 15
d13C (o/oo vs. PDB) -­‐28.5 0 d 13 C Figure 6.
-­‐29.0 -­‐29.5 -­‐30.0 -­‐30.5 -­‐31.0 Figure 6.
1 2 3 4 5 6 pH 16
d15N (o/oo vs. AIR) 0.2 0.0 d 15 N -­‐0.2 -­‐0.4 -­‐0.6 -­‐0.8 -­‐1.0 -­‐1.2 0 1 2 3 pH Figure 7.
4 5 6 17
100 Percent wax per class 90 FAL FAME ALK ph 3.5 ph 4.4 ph 5.6 80 % Wax 70 60 50 40 30 20 10 0 ph 1.6 Figure 8.
ph 2.4 18
Total wax concentration FAL+FAME+ALK 140 Concentra5on µg/ g C 120 100 80 60 40 20 0 0 Figure 9.
1 2 3 4 pH treatment 5 6 19
a)
b)
c)
d)
e)
Figure 10.
20
a)
b)
c)
d)
e)
Figure 11.
21
a)
b)
c)
d)
e)
Figure 12.
22
Long Chained (30-­‐32) FAME/FAL 37.0 R² = 0.55013 % FAME 35.0 33.0 31.0 29.0 27.0 25.0 4.0 4.5 5.0 5.5 6.0 % FAL Figure 13.
6.5 7.0 7.5 8.0 23
a)
b)
c)
d)
e)
Figure 14
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C29/31 Alkane ratio 1.8 1.6 µg/gdw 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 0 1 2 3 pH treatment Figure 15
4 5 6 25
Total Sterols 800 750 µg/gdw 700 650 600 550 500 450 400 0 Figure 16.
1 2 3 pH treatment 4 5 6 26
a)
b)
c)
d)
e)
Figure 17.
27
a)
b)
c)
Figure 18.
28
a)
b)
Figure 16.
c)
d)
e)
f)
Figure 19.
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Table 1.
Treatment
1.6 (1)
1.6 (2)
2.4 (1)
2.4 (2)
3.5 (1)
3.5 (2)
4.4 (1)
4.4 (2)
5.6 (1)
5.6 (2)
initial soil pH 11/12/13
final soil pH 11/19/13
6.6
6.5
7.1
7.2
7.2
7.1
7.3
7.3
7.3
7.4
6
6.2
7.6
7.6
7.6
7.6
7.4
7.3
7.4
7.3
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Acknowledgements
Special thanks go to my advisors Maureen Conte and JC Weber for guiding me through the
course of these 5 weeks. Fiona Jevon, Alice Carter, Sarah Nalven and Rich McHorney for their
support, Louis Kerr for sharing your knowledge of SEM, and Marshall Otter for his work done in
the Ecosystem Center’s Stable Isotope Laboratory in the MBL.
To everyone who was a part of the SES program, you are all wonderful.
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Figure 1. Total new growth after 1 week of treatment
Figure 2. Total new biomass after 1 week of treatment
Figure 3. Percent carbon by dry weight
Figure 4. Percent nitrogen by dry weight
Figure 5. Molar CN ratio
Figure 6. del 13C
Figure 7. del 15N
Figure 8. Relative Distribution of Classes
Figure 9. Total wax concentrations
Figure 10. FAL molecular distribution
Figure 11. FAL molecular distribution at a smaller scale
Figure 12. FAME molecular distribution
Figure 13. Covariance between long chain FAME and FAL
Figure 14. Alkane molecular distribution
Figure 15. Relationship between alkane C29 and C31
Figure 16. Total sterols
Figure 17. SEM images of the 5.6 pH treatment.
Figure 18. SEM images of the 3.5 pH treatment.
Figure 19. SEM images of the 1.6 pH treatment
Table 1. Measured soil pH at the initial and final day of the treatments