<b>CHARACTERIZATION OF THE PHENYLALANINE AMMONIA-LYASE (PAL) GENE FAMILY AND ITS SURROUNDING NETWORK OF REGULATION IN </b><b><i>ARABIDOPSIS THALIANA </i></b><b>WITH NOVEL CRISPR-CAS9 MUTANTS</b>

<p dir="ltr">Comprising approximately 30% of the total carbon flux in plants, the phenylpropanoid pathway generates various specialized secondary metabolites including lignin, flavonols, anthocyanins, and hydroxycinnamoyl conjugates that play key roles in defense against pathogens, oxidative stress, and UV light. The first step of the phenylpropanoid pathway, and a key regulatory point between primary and secondary metabolism, is the deamination of phenylalanine to <i>trans</i>-cinnamic acid. The enzyme responsible for this step is PHENYLALANINE AMMONIA-LYASE (PAL), which is encoded by four genes, <i>PAL1-PAL4</i>, in <i>Arabidopsis thaliana</i>. Surprisingly, previous analysis of two independent <i>pal1/2/3/4 </i>quadruple T-DNA mutants revealed that they still exhibited roughly 10% of wild-type PAL<i> </i>activity, suggesting that one or more of the <i>pal</i> T-DNA mutant alleles are leaky. To more unambiguously characterize PAL function and regulation in <i>Arabidopsis thaliana</i>, we generated exon-specific CRISPR-Cas9 true null mutants. In this study, we report that PAL1, PAL2, and PAL4<i> </i>are the major contributors toward total PAL activity, that they are the major PALs involved in soluble phenylpropanoid synthesis, and that the <i>pal1/2/4 </i>and <i>pal1/2/3/4 </i>mutants germinate and produce several small true leaves but are then developmentally arrested. Using a combination of RNA sequencing and targeted proteomics, we also report that <i>PAL </i>expression levels are largely unchanged in response to decreases in PAL activity; nevertheless, PAL2 and PAL4 protein levels are increased when<i> PAL1 </i>is disrupted, suggesting that their Kelch domain F-box protein/proteosome-mediated turnover is reduced.</p>