| Phenylpropanoid metabolism and its branch pathways produce a diverse array of compounds like suberin, lignin, phytoalexins, insect repellents and attractants and UV light protectants that play important roles during normal plant growth, development and during plant responses to biotic and abiotic stresses. We have started to dissect the complex pattern of gene regulation of this complex pathway in a model system, Arabidopsis thaliana, by isolating mutants altered in phenylpropanoid metabolite content using HPLC. We screened over 5,000 T-DNA tagged Arabidopsis plants to isolated 20 potential mutants with alterations in phenylpropanoid metabolite patterns. The mutants are classified into three major classes: (1) mutants that have lost a specific metabolite, (2) mutants that have acquired a novel metabolite and (3) mutants that show major quantitative alterations of specific metabolites. Further characterization of the two most interesting mutants, aip and C1I23, revealed that a unique morphological phenotype was associated with each mutant biochemical phenotype. For biochemical characterization of the mutants, major phenylpropanoid metabolites from wild type, and the two mutants were purified by HPLC and identified by mass spectrometry and nuclear magnetic resonance as flavonoid di- and tri-glycosides of kaempferol and quercetin. The phenylpropanoid composition found in the leaves of the mutants differed from the wild type. Organ-specific metabolite profiles of the wild type and the two mutants revealed the presence of different metabolites in different organs. The genetic analysis of the two mutants indicate that the mutations are conferred by single, nuclear, recessive genes and that aip mutant is tagged by the T-DNA. A genomic library from the aip mutants was constructed and screened to isolate potential clones with the T-DNA borders flanked by the DNA sequence from the gene of interest. To facilitate the positional cloning of the EMS mutant C1I23, the mutation was mapped to chromosome 2 by using CAPS markers. These studies serve as the first step towards elucidating the complex regulation of genes involved in the biosynthesis of phenylpropanoids in A. thaliana. Cloning of the genes will allow us to control and modify the production of phenylpropanoid derivatives in plants. |
