This study analyzed the salicylic acid methyltransferase (SAMT) protein in Asclepias curassavica milkweed. The researcher extracted RNA from A. curassavica leaf tissue, amplified the SAMT gene, cloned it into a vector plasmid, and performed assays. Analysis of the SAMT amino acid sequence showed motifs predicting preference for salicylic acid over benzoic acid. Enzyme assays using GC-MS confirmed SAMT preferentially methylated salicylic acid. Statistical analysis supported the hypothesis that SAMT amino acid sequence correlates with substrate preference.

1. Salicylic Acid Methyltransferase in Asclepias Curassavica and
Associated Protein Structure to Function Analysis
WESTON D. HILLIER
Department of Biology, Western Michigan University. 1903 West Michigan Ave. Kalamazoo, MI 49008
Abstract
All living organisms contain DNA that encodes for their development and
function; their genetic code. Simply, DNA is transcribed to mRNA, which is
translated into protein. This study looks into a protein in the class of O-
methyltransferases, salicylic acid methyltransferase (SAMT). Methyltransferase
proteins are responsible for the tastes and aromas of many plants. SAMT
proteins use salicylic acid, a cellular plant chemical defense molecule, as a
substrate. This study focuses on a specific milkweed species, Asclepias
curassavica, and it’s SAMT gene. The gene was extracted from leaf tissue,
amplified, cloned into a vector plasmid, and assayed for expression
characteristics. The data gathered on the SAMT substrate binding preference of
this individual species, A.curassavica, contributed to an overall pool of SAMT
data from a variety of plant species. I was able to create a phylogenetic tree of
protein gene sequence relationships and associated protein substrate preference
to salicylic acid (SA) and benzoic acid (BA). From our data gathered on SAMT
nucleotide sequencing and preferred substrates, I could test hypotheses on
protein structure as related to function.
Keywords: methyltransferase, milkweed, SAMT, Asclepias curassavica, salicylic acid

2. 1. Introduction
DNA encodes for development and function of all living organisms.
Transcription of DNA to mRNA, followed by translation to make protein is the
bases of all molecular biology. Proteins, the products of this biological process,
serve many roles and functions. For instance, a protein in a plant may have an
enzymatic relationship with a certain substrate and the product of that reaction
leads to an expression of pink petals, or a number of other phenotypic
expressions.
In this study, I will look at the reaction that produces methyl salicylate
(MeSA). Caffeine, theobromine in chocolate, the sent of wintergreen, and many
other lovely plant volatiles are the results of this reaction (Tieman et al. 2010).
MeSA is created by the methylation of salicylic acid (SA) by a certain enzyme in
the class of O-methyltransferase proteins (Tieman et al. 2010). Salicylic Acid
Methyltransferase (SAMT) is the protein that carries out this methylation by
binding S-Adenosyl methionine (SAM) to SA and catalyzing a reaction. Salicylic
acid is a phytohormone that contributes to pest and pathogen defense in plants
and SAM is the methyl group donor (Effmert 2005).
This study looks into the methylation of salicylic acid in the milkweed
species Asclepias curassavica. The methylation of salicylic acid allows the
compound to become volatile (Wason et al. 2013). It is believed that this volatile
methyl salicylate molecule leaves its host plant and lands on a neighboring plant
in the same population, where it is then demethylated. This increases the cellular
concentration of SA in the neighboring plant, which plays a crucial role in
activating pathogen immunity resistance (Dempsey et al 2011). MeSA and SA

3. both play a role in a plants pest and pathogen resistance response pathway
(Zhao 2010 insects).
Methyltransferases are characterized by their active sites. These active
sites have conserved motifs that always bind SAM, but show variation in binding
methylation substrate (Zubieta et al 2003). This study seeks to address two
hypotheses. The null hypothesis being there is no relationship between protein
amino acid active site sequence and an associated preference for methylation
substrate. The alternative hypothesis being there is a significant relationship
between protein amino acid active site sequence and an associated preference
for methylation substrate.
2. Methods
2.1 Bioinformatics
In order to begin this study I needed to obtain certain bioinformatics. I
used the websites <GenBank> and <1KP> to do this. GenBank was used to
retrieve nucleic acid and amino acid sequence information on the SAMT gene.
1KP was used to preform a comparison using the already known sequence for
Clarkia brewereii by running a statistical prediction algorithm called BLAST. To
begin to understand the function of my gene, I used the computer program
Phylogenetic Analysis Using Parsimony to align my protein nucleotide sequence
with other proteins that have already been studied. The program created a
phylogenetic tree of relation.

4. 2.2 RNA Extraction and PCR
When extracting for RNA, different tissues of the same plant express
different transcriptomes. This study attempted to extract RNA form both flower
and leaf tissue of milkweed using the Quick-Start Protocol with the RNeasy Plant
Mini Kit. To amplify the SAMT encoding gene I needed to run RT-PCR on the
extracted RNA. However, in order to do this I needed to design gene specific
primers for PCR. The primers needed to be between 20 and 30 base pairs long
with high G and C nucleotide content, especially crucial at the end of the primer.
These two nucleotides have three hydrogen bonds for greater stability. I had my
desired primers synthesized by Integrated DNA Technologies. My primers 1 and
2 were 24 and 23 base pairs long respectively and are listed here:
Primer 1: 5’- ATG GAA GTT GTT GAA GTT CTT CAC -3’
Primer 2: 5’- AAG CCT TCT TTT CAT GGA AAC AG -3’
The next step was to preform reverse transcriptase (RT) single-strand
cDNA synthesis on the extracted RNA using the Invitrogen First-Strand cDNA
Synthesis protocol. This protocol calls for the gene specific primers to be diluted
to 100uM solution from the primer we designed and received in dehydrated form.
I used my primer 1 because it is complementary to the sscDNA and binds to the
RNA we have for reverse transcriptase. After generating sscDNA with my desired
gene, it needed to be amplified so I could work with it. I used the Invitrogen PCR
Reaction protocol to do this. Again, I needed to use the 100uM primer solution
and dilute that further to get to 2pmol/uL. To do this, I added 49uL of water to 1uL
of our 100 uM primer solution.

5. 2.3 Gel Purification, Adenylation, and Cloning
To isolate only the desired length DNA form the PCR product I ran an
electrophoresis gel. I used a UV light box to illuminate our desired DNA and cut it
out from the gel. It is worth noting that using an adjustable intensity UV light box
on the lowest possible setting is best to reduce the possibility of mutation to the
PCR DNA product. I purified the DNA from the agarose gel using the QIAEXII
Gel Extraction Protocol.
In order for our DNA product to be properly inserted into our vector
plasmid it had to be adenylated. This acetylation process added multiple A
nucleotides to the 3’ end of the DNA, which allowed it to be inserted at the
vectors T nucleotide 5’ overhangs. This was done using the Invitrogen pTrcHis
and pTrcHis2 TOPO TA Expression Kit for Cloning and Transformation protocol.
I used 4uL of my PCR sample and 1uL of TOPO vector, for a total of 5uL.
Only 2uL of the 5uL adenylation product was used in the transformation
reaction. LB plates were created using the recipe on page 19 of the TOPO
Cloning protocol packet and then cultured. Ampicillin was added to the LB at a
1uL concentration. Plates were incubated and allowed to grow at 37oC for 24
hours. I selected 6 colonies to streak out further and produce larger cultures. I
used the same LB agar plates with ampicillin to streak the selected larger
colonies and again incubated at 37oc for 24 hours.
2.4 Screening and GC-MS
After streaking, selected colonies were grown in LB broth for use in the
QIAGEN Quick-Start Protocol QIAprep Spin Miniprep Kit. This was done to see if

6. our gene insert was transformed into the vector plasmid in the correct sense
orientation. I had to check our culture growth to be at an optimum optical
density, OD=600. Once this density was reached, I added IPTG to the culture
flask and protein expression was done at room temperature for one hour. 1ml of
50mM salicylic acid and 1mL of 50mM benzoic acid were added for equal
concentrations. Cells were pelleted in a refrigerated centrifuge and the
supernatant was collected in a new microcentrifuge tube. 4mL of hexane was
added to the solution in order to pull all non-polar products out for analysis. This
hexane phase layer was collected via pipette and analyzed using GC-MS.
To preform a statistical analysis on our hypotheses, I used a chi-square
statistical test. Observed values (O) are tested against expected values (E) for
each sequence classification and associated substrate preference. The formula
for this relationship is as follows:
In this test, I could either accept the null hypothesis, or reject the null
hypothesis by falsification and accept an alternative hypothesis. To find expected
values I used the total value for sequence type, multiplied by the value for
associated substrate preference, and divided by the grand total (See Table 1).
The degrees of freedom for this test were one.
3. Results
After running the bioinformatics, Asclepias curisavica had exact matching
sequences in the binding motifs for the SAMT protein. The results for Asclepias
curisavica were; 22-S / 57-D / 98,99-D,L / 129,130-S,F. The salicylate binding

7. motifs were; 25-Q / 145,146,147,148,149,150,151-S,S,Y,S,L,M,W,L,S / 210-L /
225,226-I,W, / 308,309,310,311-M,R,A,V. Looking at each of these motifs, there
are a few that draw significant attention. At amino acid sequence number 147
there is a Tyrosine (Y) that fills up much of the active site. This leaves any
substrate larger than salicylic acid unfavorable to bind to that site. Another
notable sequence is the –M,W,L,S- site, which is a active site that has been
studied by others and shows preference for salicylic acid. Also, my amino acid
has 150-Met, 225-Ile, 308-Met, 347-Phe, and 349-Asn, which all point to salicylic
acid substrate methylation preference.
I was able to extract RNA from both flower and leaf tissues of Asclepias
curassavica. RNA concentration extracted from flower tissue was very low, as
shown by gel electrophoresis (See figure 1). RNA concentration from leaf tissue
was higher then flowers, but still faint (See figure 2). As I proceeded with the
study using leaf tissue products, I found that the RNA extracted did not contain
any of the desired SAMT genes to be amplified by PCR (See figure 3). A final
RNA extraction from leaf tissue that had been treated with salicylic acid had a
high concentration and also had the desired SAMT gene (See figure 4). After
screening my colonies, I had one with the SAMT gene inserted in the sense
orientation (See figure 5). This sense orientation and correct base pair length
allowed me to use that colony to preform our enzyme assay and expression.
My cloning and streaking plates tuned out well. I had many
transformations of colonies onto my LB plates (See figure 6). I was able to streak
my selected colonies for gene insert orientation screening (See figure 6). The
results from my GC-MS were very promising to see. It showed that my protein

8. did show preference for methylation of salicylic acid to benzoic acid.
Comparisons were made by relative areas under both MeSA and MeBA peaks
(See figures 8 and 9). MeSA had a total area of 45,684,275 and MeBA had a
total area of only 414,517. SA was my SAMT’s preferred substrate by 100-fold.
Figure 10 shows our negative control GC-MS sample. With the negative control
we did everything the same in the protocols leading up to expression analysis,
only differing by using a vector plasmid with the SAMT insert in the antisense
orientation (See figure 10).
The statistical analysis of my data gave a chi-squared value of 12.1. With
one degree of freedom, the result was p < 0.001. This means my results were
highly significant. We can apply this to our hypotheses by stating; the variance in
our data suggests that SAMT active site amino acid sequence does correlate
with preference for certain methylation substrates. Further, our data suggests
that the amino acid sequence –MWLS- most commonly binds salicylic acid into
the SAMT active site for methylation.
4. Discussion
Certain amino acid sequences in the active sites of SAMT seem to
correlate with the methylation of certain substrates. Differences in amino acid
active site sequences leave some substrate binding pockets more apt to prefer
salicylic acid methylation to benzoic acid methylation. This substrate preference
leads to profound differences in cellular molecular composition, which may lead
to multiple different responses and functions. However, certain differences in

9. amino acid active site sequences, ones that leave very similar active site
pockets, seem to have little effect of substrate preference.
From our chi-square statistical analysis we can reject our null hypothesis
that amino acid sequence in SAMT active sites has no correlation with preferred
methylation substrates and accept our alternative hypothesis that amino acid
sequence in SAMT active sites does in fact show preference for certain
methylation substrates. More so, that a –MLWS- active site in SAMT will encode
for salicylic acid methylation substrate preference.
For future research, I would suggest to expand on the number of species
for which SAMT genes are not yet known. Gathering more data on protein amino
acid active site sequences will lead to more insight on sequence to predicted
function characteristics.
An area I am interested in further researching is the relationship between
insect herbivory loads on milkweed species and the effect on both salicylic acid
and methyl salicylate levels in plant tissues. If SA levels increase, is there an
increased activity of SAMT. Further, are there favorable planting conditions
where cellular concentrations of SA or other glycosides are increased for pest
and pathogen resistance?

10. APPENDIX
(Figure
1:
First
RNA
extraction
from
Asclepias
curassavica
leaf
tissue
agarose
gel
electrophoresis
analysis.)
(Figure
2:
Second
RNA
extraction
form
Asclepias
curassavica
flower
tissue.
Agarose
gel
electrophoresis
shows
both
flower
and
leaf
in
lanes
4
and
2
respectively.)

11.
(Figure
3:
Agarose
gel
electrophoresis
on
the
RT-­‐PCR
product.
My
sample
was
loaded
in
well
3,
with
no
product
visible.)
(Figure
4:
Agarose
gel
electrophoresis
of
RNA
extraction
from
salicylic
acid
treatment
of
Asclepias
curassavica
leaf
tisse.
My
sample
was
loaded
into
well
4,
with
a
strong
concentration
of
product.)

12.
(Figure
5:
Agarose
gel
electrophoresis
image
of
my
RT-­‐PCR
product
using
my
salicylic
acid
treatment
RNA.
I
loaded
two
wells,
in
the
brackets.)
(Figure
6:
These
are
my
plated
colonies.
The
top
four
are
my
RT-­‐PCR
product
that
has
been
put
into
the
vector
plasmid
and
grown
on
LB
agar
ampicillin
plates.
The
bottom
plate
is
of
six
colonies
that
were
chosen
to
be
streaked.)

13.
(Figure
7:
This
is
the
gel
electrophoresis
image
of
the
cloning
test.
I
took
three
of
my
streaked
c…………….)
(Figure
8:
This
shows
the
summary
of
our
GC-­‐Mas
Spec.
Peak
1
corresponds
to
MeBA
and
peak
2
corresponds
to
MeSA.
Each
molecule
is
given
the
time
it
appeared
along
with
the
total
area
under
each
peak.)

14.
(Figure
9:
This
is
the
GC-­‐MS
overall
results.
Both
benzoic
acid
and
salicylic
acid
peaks
are
shown
in
relation
to
another
in
abundance
and
time
released.)
(Figure
10:
This
is
the
GC-­‐MS
results
of
the
negative
control.
Our
negative
control
was
an
anti-­‐sense
gene
plasmid
insert.)

15.
(Figure
11:
This
is
the
phylogenic
tree
from
our
SAMT
gene
sequencing
and
protein
assay
expression.
Substrate
methylation
preferences
to
salicylic
acid
and
benzoic
acid
are
given
to
the
right
of
each
species.)
(Table
1:
The
table
below
shows
the
values
for
my
chi-­‐squared
statistical
test.
Green
numbers
represent
observed
values
and
red
numbers
represent
expected
values.)

16. BIBLIOGRAPHY
Dempsey, D.A., et. al. (2011). Salicylic Acid Biosynthesis and Metabolism. The
American Society of Plant Biologists.
Effmert, U., et. al. (2005). Floral benzenoid carboxyl methyltransferases: From in vitro to
in planta function. Phytochemistry. 66: 1211-1230.
Tieman, D., et al. (2010). Functional analysis of a tomato salicylic acid methyl
transferase and its role in synthesis of the flavor volatile methyl salicylate.
Blackwell Publishing Ltd, The Plant Journal, 62: 113–123.
Wason, E.L., et. al. (2013). A Genetically-Based Latitudinal Cline in the Emission of
Herbivore-Induced Plant Volatile Organic Compounds. Journal of Chemical
Ecology. 39: 1101–1111.
Zhao, N., et. al. (2010). Biosynthesis and emission of insect-induced methyl salicylate
and methyl benzoate from rice. Plant Physiology and Biochemistry. 48: 279-287.
Zubieta, C., et. al. (2003). Structural Basis for Substrate Recognition in the
Salicylic Acid Carboxyl Methyltransferase Family. Plant Cell, 15(8): 1704–1716.