| Plants cannot escape from herbivory and other environmental stresses including cold and drought, and have evolved sophisticated defense systems to survive. Improving food production has become one of the most urgent problems facing humankind with 7 billion population and limited land available. Plants also serve as the base of the earth's sustainable fuel supply, and synthesize a diverse suite of natural compounds that help defend them against stress, but also are potential pharmaceutics. Improving our understanding of plant defense systems is a key factor in using plants as natural resources to provide solutions to these problems. To defend themselves, plants synthesize oxidized fatty acids, or oxylipins, to regulate gene expression in response to stress. In response to wounding and certain stresses, many plants synthesize the cyclopentenone oxylipin 12-oxo-phytodienoic acid (OPDA) as a precursor of the master regulatory hormone jasmonic acid (JA). JA is then conjugated to isoleucine in cytoplasm to produce the universal defense gene regulator JA-isoleucine. OPDA has been shown to be an independent but not fully understood metabolite that regulates plant defenses. Reactive electrophiles such as OPDA are subject to conjugation to the tripeptide glutathione. This process is catalyzed by an assortment of glutathione transferase enzymes, and is expected to deactivate the biological functions of electrophiles. However, understanding of the functions of specific GSTs (>50 in model plant Arabidopsis from sequenced genome), particularly in plants, is limited. The research described in this dissertation has aimed to reveal biological functions of GSTs in defense response to mechanical wounding in Arabidopsis. The basic strategy has combined information about GST protein levels with profiling of metabolites in a GST knockout mutant to correlate metabolic phenotypes with genotypes, for deduction of gene functions. 8 GSTs were identified in wild type Arabidopsis leaves, and one of them, AtGSTU5, was shown to be highly accumulated after mechanical wounding. Several Arabidopsis knockout mutants, including AtGSTu5, were grown for wounding experiments and phenotype assessment using non-targeted metabolite profiling. The glutathione conjugate of OPDA was quantified and shown to be accumulated after mechanical wounding in leaves of wild type and other tau-family GST knockout mutants of Arabidopsis, but not in the knockout mutant AtGSTu5. This finding suggests that AtGSTU5 is responsible for in vivo glutathione conjugation of OPDA. A new LC-TOF-MS protocol was developed to explore the range of endogenous glutathione conjugates in extracts of Arabidopsis leaves. A family of novel glutathione conjugates is also discovered and proposed to be derived from OPDA-containing galactolipids. In the identification and quantitation of plant oxylipins, tandem mass spectrometry (MS/MS) data are often the primary source of metabolite structural information, but ion fragmentation pathways are not well understood for negative ions. This dissertation describes a novel fragmentation mechanism in negative mode involving charge-directed hydride migration for OPDA and its lower homolog dinorOPDA. This mechanism has potential to guide structure elucidation of unknown oxylipins. For other cyclopentenone prostaglandins with greater degrees of unsaturation, however, fragmentation behavior differed. Relative amounts of specific product ions distinguished prostaglandins with identical side chains but opposite cyclopentenone ring orientations. |
