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Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the deamination of phenylalanine, which is the initial step in the biosynthesis of phenylpropanoids. It serves as a crucial enzyme that facilitates the transfer of carbon from primary to secondary metabolism in plants. Duckweed is regarded as a promising chassis plant in synthetic biology research and application, due to its being rich in secondary metabolites and other advantages. The genes encoding PAL in <i>Spirodela polyrhiza</i> (L.) Schleid, the giant duckweed, were investigated in this study. Three <i>SpPAL</i> genes (<i>SpPAL1–SpPAL3</i>) were identified and cloned. All of them were successfully expressed in <i>E. coli</i>, and their recombinant proteins all showed PAL activity. In addition, SpPAL1 and SpPAL2 proteins could also utilize tyrosine as substrate, although the activity was low. A qRT-PCR analysis demonstrated that the expression of <i>SpPAL3</i> was most pronounced in young fronds. It was found that the expression of <i>SpPAL1</i> and <i>SpPAL3</i> was significantly induced by MeJA treatment. Overexpression of <i>SpPAL3</i> in <i>Lemna turionifera</i> inhibited the growth of fronds and adventitious roots in the transgenic plants, indicating the importance of <i>SpPAL3</i> in duckweed besides its involvement in the secondary metabolism.