Overexpression of the Glycyrrhiza uralensis Phenylalanine Ammonia-Lyase Gene GuPAL1 Promotes Flavonoid Accumulation in Arabidopsis thaliana

Phenylalanine ammonia-lyase (PAL) serves as a pivotal regulatory enzyme at the initial branching point of the phenylpropanoid pathway, exerting a profound influence on downstream reactions essential for flavonoid biosynthesis. <i>Glycyrrhiza</i> species are important medicinal plants and provide plenty of roots as raw materials for further utilization, with the components of glycyrrhizic acid and flavonoids as two major active ingredients. However, functional studies of the <i>PAL</i> genes in the medicinal <i>Glycyrrhiza</i> species remain limited. In this study, we identified seven <i>PAL</i> family genes from each of the three medicinal <i>Glycyrrhiza</i> species, <i>Glycyrrhiza uralensis</i> Fisch., <i>G. inflata</i> Bat., and <i>G. glabra</i> L., and comprehensively analyzed their phylogenetic relationships, gene structures, motif distributions, and promoter <i>cis</i>-elements. Gene expression profiling revealed that <i>PAL1</i> is highly expressed in roots and significantly induced by drought and salt stresses. We further selected <i>G. uralensis GuPAL1</i> for functional investigation in <i>Arabidopsis</i>. <i>GuPAL1</i>-overexpression lines (<i>GuPAL1</i>-OE) demonstrated significant enhancements in plant growth, flavonoid accumulation, and hormone levels in <i>Arabidopsis thaliana</i>. Conversely, the <i>Atpal1</i> mutant plants displayed marked reductions in these traits, while the transgenic lines of <i>GuPAL1-</i>OE in the <i>Atpal1</i> mutant (<i>Atpal1</i>/<i>GuPAL1</i>) recovered to the normal phenotypes similar to wild type (WT). Transcriptomic analysis of the <i>GuPAL1</i>-OE plants compared to WT demonstrated that several key genes in the phenylpropanoid and flavonoid metabolic pathways (<i>4CL</i>, <i>CCoAOMT</i>, <i>CAD</i>, <i>POD</i>, <i>F3H</i>, <i>FLS</i>) were significantly enriched, suggesting that <i>GuPAL1</i> may promote plant growth and flavonoid biosynthesis by regulating diverse cellular functions, metabolic pathways, and associated gene expressions. These findings highlight the functional importance of <i>GuPAL1</i> in flavonoid biosynthesis, and provide valuable insights into the molecular mechanisms underlying the medicinal properties of <i>Glycyrrhiza</i> species.