Fall15 UROP Poster
1
Camalexin Production in Arabidopsis thaliana is Regulated by the MYB51 and MYB122 Transcription Factors Lawrence Bandoni 1 , John Celenza 2 Department of Biology, Boston University 1 , 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 r o 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 1 2 3 4 5 6 7 8 9 10 Myb34 Myb51 Myb122 Myb34/51 Myb34/122 Myb51/122 Myb TKO Columbia Amount of Camalexin (ng camalexin per mg tissue) Lineage A 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Myb34 Myb51 Myb122 Myb34/51 Myb34/122 Myb51/122 Myb TKO Columbia Amount of Camalexin (ng camalexin per mg tissue) Lineage B 0 1 2 3 4 5 6 7 Myb34 Myb51 Myb122 Myb34/51 Myb34/122 Myb51/122 Myb TKO Columbia Amount of Camalexin (ng camalexin per mg tissue) 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
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Transcript of Fall15 UROP Poster
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
1
2
3
4
5
6
7
8
9
10
Myb34 Myb51 Myb122 Myb34/51 Myb34/122 Myb51/122 Myb TKO Columbia
Am
ount
of C
amal
exin
(ng
cam
alex
in p
er m
g tis
sue)
Lineage
A
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Myb34 Myb51 Myb122 Myb34/51 Myb34/122 Myb51/122 Myb TKO Columbia
Am
ount
of C
amal
exin
(ng
cam
alex
in p
er m
g tis
sue)
Lineage
B
0
1
2
3
4
5
6
7
Myb34 Myb51 Myb122 Myb34/51 Myb34/122 Myb51/122 Myb TKO Columbia
Am
ount
of C
amal
exin
(ng
cam
alex
in p
er m
g tis
sue)
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)
AcknowledgementsUROP
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 ExtractionCamalexin 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
