As Arabidopsis thaliana ages it becomes more resistant to virulent Pseudomonas syringae pv. tomato (Pst) bacteria. This is known as age-related resistance (ARR). ARR is associated with flowering and intercellular accumulation of salicylic acid (SA). A microarray experiment identified a number of jasmonic acid/ethylene (JA/ET)-associated genes whose expression was up-regulated during ARR. This thesis explores the role of JA/ET signaling during ARR by characterizing the role of two JA/ET-associated No Apical Meristem Cup-shaped Cotyledons (NAC) transcription factors: ANAC055 and ANAC092. Analysis of nae single and double mutants suggests that the NACs play nonredundant roles during ARR. The partial ARR defect of anac092 is followed one week later by an enhanced ARR response, and this is associated with a delay in flowering. Furthermore, mature 35S:ANAC092 exhibits increased susceptibility to Pst. Collectively, this data suggests that ANAC092 is a negative regulator of ARR and it contributes to positive regulation of flowering and the onset of ARR. The late flowering mutant luminidependens1 also exhibits a partial ARR defect, suggesting that the autonomous flowering pathway contributes to ARR. Gene expression data suggests that ANAC055 and ANAC092 regulate expression of some JA/ET-associated genes during ARR. The JA/ET signaling mutant ethylene insensitive2 (ein2) exhibits a partial ARR defect and reduced expression of ANAC055 and ANAC092, suggesting that EIN2 is a positive regulator of expression of ANAC055 and ANAC092 during ARR. Phytohormone analyses reveal that JA accumulates to similar levels in young and mature wild-type plants after inoculation with Pst, suggesting that increased expression of some JA/ET-associated genes in mature compared to young plants after inoculation with Pst is not associated with elevated levels of JA. This thesis contributes to our understanding of ARR by identifying some components of the NAC pathway, exploring the relationship between flowering and ARR, and conducting some phytohormone analysis. / Thesis / Doctor of Philosophy (PhD)

Many economically important crop systems exhibit an Age-Related Resistance (ARR) response whereby mature plants become resistant to pathogens they were susceptible to when younger. The signaling pathways and mechanisms of ARR have not been well studied. Arabidopsis displays ARR in response to P. syringae pv tomato (Pst). Several studies provide evidence that intercellular salicylic acid (SA) accumulation is required for ARR and SA acts as a direct antimicrobial agent to limit bacterial growth and biofilm-like aggregate formation. SA accumulation mutants are ARR defective; however, a modest level of resistance is occasionally observed leading to the hypothesis that other compounds contribute to ARR as antimicrobial agents. Previous studies demonstrated that CYP71A13 (a key enzyme in indolic biosynthesis) is expressed during the ARR response. I demonstrated that CYP71A12 functionally compensated for CYP71A13 during ARR, as cyp71a12/cyp71a13-1 mutants were consistently ARR-defective compared to their respective single mutants. I demonstrated that dihydrocamalexic acid (DHCA) accumulated in intercellular washing fluids (IWFs) collected from plants during the ARR response using high resolution mass spectrometry-based profiling. DHCA was detected in IWFs collected from wild-type ARR-competent plants and, was absent in IWFs from ARR-incompetent cyp71a12/cyp71a13 mutants. In vitro DHCA antimicrobial activity against P. syringae was not observed, but exogenous infiltration of DHCA into the leaf intercellular space restored ARR in cyp71a12/cyp71a13 mutants Unlike SA which exhibits direct antimicrobial activity against P. syringae, DHCA does not and instead may affect pathogen virulence in other ways. My research provides evidence that intercellular DHCA contributes to ARR in response to P. syringae in Arabidopsis. Understanding the genes and metabolites contributing to ARR will provide useful information for future crop breeding and genetic modification that will mitigate agricultural losses due to disease. / Thesis / Master of Science (MSc) / During Age-Related Resistance (ARR), mature plants including some crop plants become resistant to pathogens they were susceptible to when younger. How ARR works is poorly understood. My objective was to identify potential antimicrobial metabolites contributing to ARR in Arabidopsis against the bacterial pathogen Pseudomonas syringae. Genetic analyses combined with mass-spectrometry based metabolite profiling demonstrates that two cytochromes P450, CYP71A12 and CYP71A13 contribute to ARR. My research provides evidence that DHCA accumulates in the leaf intercellular space in ARR-competent plants, where it may act to inhibit the bacterial infection process. DHCA has low antimicrobial activity against P. syringae suggesting its mechanism of action is not directly antimicrobial. Importantly, application of DHCA to the leaf intercellular space of cyp71a12/cyp71a13 restored ARR, confirming that DHCA contributes to ARR in Arabidopsis. Understanding ARR will provide useful information for future crop breeding and genetic modification that will mitigate agricultural losses due to disease.