Research Assistant Purdue University West Lafayette, Indiana
Body of Abstract: The phenylpropanoid pathway generates various specialized secondary metabolites that play key roles in defense against pathogens, oxidative stress, and UV light. The first step of the phenylpropanoid pathway is the deamination of phenylalanine to trans-cinnamic acid. The enzyme responsible, phenylalanine ammonia-lyase (PAL), is encoded by four genes PAL1-PAL4 in Arabidopsis thaliana. Previously, T-DNA mutant lines of PAL had been generated. Surprisingly, analysis of two independent pal1/2/3/4 mutants revealed that they exhibited 10% of wild-type PAL activity, suggesting that one or more of the pal T-DNA mutant alleles are leaky. As a result, the previous work on higher level pal mutants may not faithfully represent the roles of each PAL gene in phenylpropanoid metabolism. To characterize PAL function and regulation in Arabidopsis thaliana, we have generated exon-specific CRISPR-Cas9 mutants and have observed various differences that distinguish our CRISPR mutants from the T-DNA mutants. PAL assays performed on our CRISPR pal lines agree with previous findings that PAL1 is the major isoform contributing to total PAL activity. CRISPR pal1/2 line showed decreased rate of germination and growth compared to Col-0 and the previously reported pal1/2 T-DNA mutant. CRISPR pal1/2 and pal1/2/3 plants produce yellow seeds, something previously reported in the T-DNA lines as well. Interestingly, our findings suggest that both PAL1 and PAL2 need to be knocked-out in the same background to produce the yellow seeds and that a single copy of wild-type PAL1 allele is enough to prevent this phenotype. Additionally, CRISPR pal1/2/3 lines exhibited stunted growth and sterility despite previously being reported to be fertile. Although the project is still in its early stages, our novel CRISPR pal mutants as well as our experience in the phenylpropanoid pathway provides us with an advantageous position to fully characterize and elucidate the function and regulation of the PAL family in Arabidopsis thaliana.