Phytohormones in Arabidopsis thaliana during Pseudomonas syringae infection.

Open Access
- Author:
- Seidl-Adams, Irmgard Helga
- Graduate Program:
- Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- July 31, 2006
- Committee Members:
- John Charles Schultz, Committee Chair/Co-Chair
John Edward Carlson, Committee Member
Hong Ma, Committee Member
Simon Gilroy, Committee Member - Keywords:
- Pseudomonas syringae pv tomato
Salicylic acid
Jasmonic acid
auxin
coronatine
CYP79B2B3
defense signaling - Abstract:
- ABSTRACT Coronatine is often referred to as a JA mimic. As a JA mimic coronatine could interfere with defense signaling during Pseudomonas syringae pv tomato (P.s. pv tomato) infection. We investigated possible effects of coronatine on defense signaling by measuring Salicylic Acid (SA), Jasmonic Acid (JA), and auxin (IAA) concentrations in Columbia wildtype (Col WT) Arabidopsis thaliana (A. thaliana) plants infected with virulent P.s. pv tomato DC3000; and mutant bacterial strains DC3000 AK87 and DC3000 DS29, which are incapable of synthesizing coronatine (COR-) or either of its subunits, coronafacic or coronamic acid. Similarly, hormone concentrations were measured in Col WT and coronatine insensitive (coi1) mutant Arabidopsis plants infected with WT DC3000 bacteria. Hormone measurements were conducted simultaneously with gas chromatography and subsequent detection with mass spectrometry (GC-MS). Bacterial growth and decreasing leaf weight as a measure of disease progression at all sampling time points were also monitored. From the inoculation with COR- DC3000 bacteria I conclude that coronatine suppresses SA concentration increases, augments IAA concentration increases, and has no effect on JA concentration increases at later phases of infection. From the inoculation of coi1 and Col WT plants with WT DC3000 bacteria I conclude that coronatine is not a JA mimic. Coronatine suppresses SA concentration increases at least in part independently of functional COI1 because SA concentration increases during infection with COR- DC3000 strains were significantly larger than in coi1 mutants at the later time point measured in the infection. Furthermore IAA concentration increases are not affected by COI1 signaling. Therefore the augmenting effect of coronatine on IAA accumulation must happen in a COI1-signaling independent manner. While the interaction of SA and JA is well documented and studied, the role of IAA in defense responses has not received a lot of attention. Knowing the origin of IAA concentration increases opens avenues to address the role and regulation during pathogen infection. Therefore I investigated the origin of auxin during P.s. pv tomato infection. I tested whether IAA concentration increases are due to synthesis from tryptophan via the action of CYP79B2/B3, hydrolysis from IAA-amido conjugates or transport into inoculated leaves. IAA was at least in part newly synthesized via the action of CYP79B2 and/or CYP79B3; the contribution of hydrolysis from IAA-amido conjugates was inconclusive; and transport did not play a role in the accumulation of IAA. Disease progression in the cyp79b2/b3 double mutants was altered, JA accumulation was significantly reduced, while SA accumulation was unaltered. These findings suggest that IAA accumulation affects JA accumulation. The cyp79b2/b3 double mutant should allow studying further the role of IAA on symptom development and pathogen growth. The third section of this thesis is a theoretical treatise about the validity of real-ime PCR quantification methods commonly used. The implicit and explicit assumptions of the standard curve model and two models using exponential or logistic functions to describe the progression of individual reactions were analysed, and tested whether they calculate relative differences between template concentrations accurately. I developed an Excel Workbook-based method, which allows determining semi-automatically the range of exponential amplification in individual real-time PCRs. I concluded that when high accuracy is necessary the standard curve method is still the method of choice to determine relative template concentrations. Conditions under which the shortcomings of the considered methods become most pronounced are discussed.