BIOS 5260 Term Paper

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

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

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.

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.

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

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

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

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

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?

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.)  

 (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.)  

 (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.)  

 (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.)  

 (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.)        

 (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.)  

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.