Salicylic acid (SA) is involved in mediating defense against biotrophic pathogens. The current knowledge of the SA-mediated signaling pathway and its effect on the transcriptional regulation of... Show moreSalicylic acid (SA) is involved in mediating defense against biotrophic pathogens. The current knowledge of the SA-mediated signaling pathway and its effect on the transcriptional regulation of defense responses are reviewed in this thesis. PR-1 is a marker gene for systemic acquired resistance (SAR) and we have shown that the plant-specific transcription factor WRKY50 binds to the Arabidopsis PR-1 promoter at two positions close to binding sites of TGA proteins, bZIP class transcription factors previously shown to be important for PR-1 expression. We found that WRKY50 interacts with TGAs and together they synergistically activate PR-1 expression. In addition, a number of W-boxes in the PR-1 promoter have been shown to be also important for expression. We have shown that WRKY28 is able to bind to these W-boxes and to activate PR-1 gene expression in protoplasts. We also describe the effects of overexpression of the WRKY50 and WRKY28 proteins in transgenic plants. High constitutive expression of the WRKY50 gene resulted in accumulation of PR-1 mRNA when the plants were treated with SA. The overexpression of WRKY50 and WRKY28 had no clear effect on the plants__ resistance to the biotrophic pathogens, and neither was this the case with WRKY50 T-DNA knockout plants. To investigate the WRKYs__ influence on metabolite composition, a 1H NMR spectroscopy-based metabolomic approach was applied. The WRKY50 overexpression plants contained two- to three-fold more sinapic acid derivatives. This indicates a possible involvement of WRKY50 on metabolite production, in particular of hydroxycinnamates such as sinapic acid. As these compounds are components of lignin, this may point to a role of WRKY50 in stress-induced lignin modification. Show less
Jasmonic acid (JA) is a plant hormone that plays an important role in defense against wounding, insects and microbial pathogens. In defense responses against microbial pathogens JA acts... Show moreJasmonic acid (JA) is a plant hormone that plays an important role in defense against wounding, insects and microbial pathogens. In defense responses against microbial pathogens JA acts synergistically with the stress hormone ethylene. ORA59, belonging to the plant-specific class of AP2-domain transcription factors, is the main regulator of JA/ethylene-responsive defense gene expression in the model plant species Arabidopsis thaliana. The aim of the research described in this thesis was to study how the activity of ORA59 is regulated by JA. Studies on the promoter of the defense gene PDF1.2 showed that ORA59 regulates gene expression by interaction with GCCGCC motifs. Studies of ORA59 at the protein level indicated that JA controls ORA59 activity by promoting the nuclear localization and stabilization of the protein. Based on the findings it was postulated that there is an F-box protein that targets ORA59 for degradation, and a repressor protein that sequesters ORA59 in the cytoplasm. A protein interacting with ORA59 was identified by yeast two-hybrid screening. Further studies showed that this protein acts as a repressor of ORA59 by retaining it in the cytoplasm, which leads to fine-tuning of basal resistance against pathogens. Show less
The salicylic acid (SA) signaling pathway triggered by attack of biotrophic pathogens leads to broad spectrum resistance against a plethora of pathogenic fungi, bacteria and viruses and is known as... Show moreThe salicylic acid (SA) signaling pathway triggered by attack of biotrophic pathogens leads to broad spectrum resistance against a plethora of pathogenic fungi, bacteria and viruses and is known as systemic acquired resistance (SAR). One of the hallmarks of SAR is the accumulation of PR proteins and the induced expression of the PR-1 gene is often used as a marker for SAR. In this thesis we describe the characterization of a WRKY-class transcription factor (NtWRKY12) that specifically binds to the sequence TTTTCCAC. This sequence differs significantly from the consensus WRKY protein binding site TTGAC[C/T] (W-box), and was designated as the __WK-box__. The activation of PR-1a has always been proposed to occur mainly via TGA-factors. We show that NtWRKY12 can bind to TGA2.2 and has a more essential role in PR-1a activation. Using bioinformatic analysis various candidate genes have been prospected to have a role in regulation of the SA, jasmonate (JA) and ethylene (ET) biosynthesis or signaling pathways. The connection between AtWRKY28 and ICS1 was further studied. In protoplasts AtWRKY28 can activate ICS1 promoter::GUS reporter constructs and endogenous ICS1 mRNA levels. Using EMSA assays and ChIP experiments the bindingsite has been determined in vitro and confirmed in vivo. Show less
Plants defend themselves against stress, including pathogen or herbivore attack, via biosynthesis of defense proteins and of protective compounds called secondary metabolites. Stress induces these... Show morePlants defend themselves against stress, including pathogen or herbivore attack, via biosynthesis of defense proteins and of protective compounds called secondary metabolites. Stress induces these responses via a complex signal transduction network with jasmonic acid (JA) and related compounds as major players. In some defense responses JA acts synergistically with the stress hormone ethylene. It is largely unknown how JA induces expression of defense genes. It is also not well understood how the synergistic interaction between JA and ethylene is brought about. Previous research has identified several genes encoding AP2/ERF-domain transcription factors, called ORAs, whose expression is rapidly induced by JA in Arabidopsis. In this thesis, the function of several ORAs is addressed and shows that certain ORAs, by regulating the expression of defense-related genes, play major roles in JA signaling and defense. Show less
