1. Camalexin Production in Arabidopsis thaliana is Regulated by the
MYB51 and MYB122 Transcription Factors
Lawrence Bandoni1, John Celenza2
Department of Biology, Boston University1, Boston, MA
Camalexin and indolyl-3-methyl indolic
glucosinolate (I3M) are produced in Arabidopsis thaliana as
defense compounds. I3M is produced in response to tissue
damage and thus it is believed that I3M is produced to prevent
herbivory. Camalexin, on the other hand, is produced in
response to microbial infection, and is produced in order to deter
spread of the infection. Both camalexin and I3M are tryptophan
(Trp) derivatives, and their production is dependent on
expression of the CYP79B2 and CYP79B3 genes, which are
responsible for converting tryptophan to indole-3-acetaldoxime
(IAOx). IAOx is a branch point that separates the production of
camalexin from that of I3M. Expression of CYP79B2 and
CYP79B3 are regulated by three closely related Myb transcription
factors called MYB34, MYB51, and MYB122.
Recent work suggests that MYB34 and MYB51,
but not MYB122, are sufficient for the production of I3M. This
past summer, my goal was to determine which of these Myb
family transcription factors are required for camalexin production.
To achieve this, single and multiple myb mutant genotypes were
grown in liquid media for ten days after which the plants were
treated for three days with silver nitrate, methyl jasmonate, or
Flg22, known elicitors of camalexin. Camalexin was then
quantified by HPLC and it was found that that MYB51 and
MYB122, but not MYB34 are necessary for camalexin production.
These results, combined with the previous IG results, suggest
that the different MYB genes have evolved to have specialized
roles in producing distinct Trp-derived defense compounds in
response to different environmental stresses.
The production of natural defense compounds
such as camalexin is vital to the development and survival of
many plants. Due to its genomic similarity to many other plants
including crops that humans rely on for food, and its relatively
small genome, Arabidopsis thaliana has become a model
organism in plant biology. Therefore, our results do not only help
us build a better understanding of the biochemical pathways by
which most plants produce defense compounds, but they also
have the potential to help in designing crop plants that are more
adaptable to environmental stresses.
Arabidopsis thaliana is a small, flowering weed
native to Europe and Asia. Due to its small size, small genome
(about 135Mbp), and rapid life cycle, Arabidopsis thaliana is
perhaps the most widely-studied model organism in plant biology.
Much like animals, Arabidopsis thaliana has evolved mechanisms
to avoid predation and disease. While animals often use the
fight-or-flight pathway to avoid predation, plants can neither fight
nor flee from their predators, and are thus limited to producing
certain natural defense compounds in order for their species to
survive. Capsaicin, for example, is the compound produced by
hot peppers that makes them “spicy.” In reality, the capsaicin
pathway likely evolved in order to avoid predation. Similarly,
Arabidopsis produces a variety of natural defense compounds,
two of which are indolyl-3-methyl glucosinolate (I3M) and
camalexin. I3M is produced in response to tissue damage and
thus it is believed that I3M is produced to prevent herbivory.
Camalexin, on the other hand, is produced in response to
microbial infection, and is produced in order to deter spread of
the infection. Both of these compounds are derivatives of the
amino acid tryptophan, and their production is dependent on
expression of the CYP79B2 and CYP79B3 genes, which are
responsible for converting tryptophan to indole-3-acetaldoxime
(IAOx). IAOx is the branch-point intermediate between tryptophan
and I3M and camalexin. Expression of CYP79B2 and CYP79B3
is regulated by three closely related Myb family transcription
factors called MYB34, MYB51, and MYB122. Recent work
suggests that MYB34 and MYB51, but not MYB122 are sufficient
for proper regulation of production of I3M. Because of this result,
my goal this past summer was to determine the combination of
MYB genes necessary for production of camalexin. Arabidopsis
thaliana is a member of the Brassicaceae family, meaning that it
is genetically similar to crop plants such as broccoli, cauliflower,
and cabbage. Therefore, the findings of this study not only shed
light on the underlying biochemical pathways of natural defense
compound production, but they also have the potential to help in
designing crop plants that are more adaptable to environmental
stresses.
u
Introduction
Abstract
ro
p
• fast growing rate 6-8 weeks from
seed to seed
• self-fertile diploid makes for simple
genetics
• easily transformable for genetic
modification
• 135 Mb genome on 5 chromosomes
of and encoding 26,000 genes
0
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10
Myb34 Myb51 Myb122 Myb34/51 Myb34/122 Myb51/122 Myb TKO Columbia
AmountofCamalexin(ngcamalexinpermgtissue)
Lineage
A
0
0.2
0.4
0.6
0.8
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1.2
1.4
1.6
1.8
2
Myb34 Myb51 Myb122 Myb34/51 Myb34/122 Myb51/122 Myb TKO Columbia
AmountofCamalexin(ngcamalexinpermgtissue)
Lineage
B
0
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Myb34 Myb51 Myb122 Myb34/51 Myb34/122 Myb51/122 Myb TKO Columbia
AmountofCamalexin(ngcamalexinpermgtissue)
Lineage
C
Arabidopsis thaliana Results
Conclusion
Tryptophan Metabolism in A. t.
References
Methods
• For silver nitrate and methyl jasmonate treatment,
camalexin produced in myb51 myb122 double knockout
plants (or the triple mutant) was significantly less than in
other single mutant or double mutant combinations.
• When treated with Flg22, the results were anomalous in that
the triple mutant produced the most and wild type produced
the least.
• Our results suggest that MYB51 and MYB122 are sufficient
to convert IAOx to camalexin.
• MYB34 is not necessary for camalexin production while it is
important for IG production.
• These results, combined with the previous IG results, show
that the different MYB genes have evolved to have
specialized roles in producing distinct Trp-derived defense
compounds in response to different environmental stresses.
Future Directions
• Further investigate abnormal responses to Flg22 stimuli.
• Investigate other transcription factor families in the IG and
Camalexin pathways (ex. MYC2, MYC3, MYC4)
Acknowledgements
UROP
Celenza Lab
• Brad Hogan
• Sanda Zolj
• Dylan Vaughan
• Shruthi Rengarajan
• Nahomie Rodriguez-Sastre
• Drew Bergman
• Cindy Shi
• The graphs in each panel show the average amount of
camalexin produced by each mutant (the sum of camalexin
found in tissue and media extractions). The corresponding
error bars show the standard error in camalexin production
among each group of mutants.
• Panel A shows the production of camalexin in plants treated
with silver nitrate. myb51 myb122 double mutants (and the
triple mutant) produced significantly less camalexin than all
other lines, except myb51 single knockouts. Additionally,
camalexin production in the triple knockout was not
significantly different from that of the myb51 122 double
mutants.
• Panel B shows the production of camalexin in plants treated
with methyl jasmonate. myb51 122 double mutants
produced significantly less camalexin than all other lines.
Camalexin production in these plants was similar to that of
plants treated with silver nitrate.
• Panel C shows the production of camalexin in plants treated
with Flg22. All single and double knockout plants produced
similar amounts of camalexin. Additionally, triple knockouts
produced significantly more camalexin than all other lines,
and wild-type plants produced significantly less camalexin
than other plants.
• Hogan, B. J. (2013). Partially redundant tryptophan
synthase and MYB transcription factor genes regulate
indolic defense compound synthesis in Arabidopsis
thaliana.
• In order to determine which gene or combination of genes
regulates camalexin production, A. thaliana plants were
grown in vitro in a plant nutrient solution (PNS) for ten days.
During the final three days of growth, one group of plants
was treated with PNS containing 0.1 mM Silver Nitrate,
another with PNS containing 0.02 mM Methyl Jasmonate,
and the third with PNS containing 0.1 µM of the flagellar
protein Flg22.
• Each treatment contained 32 wells of 8-20 plants each,
which included four of each of the following knockouts:
myb34, myb51, myb122, myb34 myb51, myb34 myb122,
myb51 myb122, myb34 myb51 myb122, and wild type
(Col).
• Once each group of plants had finished growing, a
methanol and chloroform extraction was done on each
plant and the media in which it was grown in order to
extract the total camalexin produced.
• Chromatography via HPLC was performed on the extract
from each plant to quantify the relative amount of camalexin
the plant produced (ng Camalexin per mg tissue).
Camalexin Extraction
Camalexin was extracted separately from plant tissue and the
growth media as follows:
• Tissue samples were removed from each well, dried,
weighed, frozen on dry ice, crushed, submerged in 0.5mL
80% methanol, then heated at 80ºC for 15 minutes. Media
was extracted with an equal volume of 80% methanol and
then evaporated to 0.5 ml. Tissue and media methanol
extracts were then treated the same.
• Methanol extracts were extracted with an equivalent
amount of chloroform.
• Chloroform extracts evaporated to dryness re-suspended in
300µL methanol and filtered.
• The media and plant extracts were run separately on the
HPLC and amounts of camalexin added together.
HPLC Analysis
• Both plant tissue and media extractions were run on a C-18
column with a mobile phase of 40% methanol for a run time of
26 minutes per sample.
• In the chromatogram of each sample, the integral of the
camalexin peak was taken (this peak appeared at about 12
minutes in the chromatogram based on its UV absorbance
spectrum compared to authentic camalexin).
• The integral of the camalexin peak was used to calculate the
amount of camalexin by comparison to a known amount.
This figure shows the pathway by which A. thaliana metabolizes
tryptophan into various natural defense compounds. Recent work
has shed light on the way Myb family transcription factors
regulate production of Indole glucosinolates. My research
focuses on the production of camalexin (as indicated by the red
arrow)
The camalexin peak in the chromatogram
A flow chart of the camalexin extraction method