*2.2. Analysis of the Gene Structures, Conserved Motifs, and Phylogenetic Relationships of* PLP\_deC *Genes*

To clarify the evolutionary relationships among the *PLP\_deC* genes, we compared the PLP\_deC proteins from *Arabidopsis thaliana (A. thaliana)*, *Oryza sativa (O. sativa)*, *D. o*ffi*cinale* and *P. equestris*. We used the maximum likelihood (ML) method to construct a phylogenetic tree using IQ-TREE software. As shown in Figure 1, the 42 *PLP\_deC* genes could be divided into three subfamilies: GAD, HDC, and aromatic-L-AAD. The *PLP\_deC* genes in *D. o*ffi*cinale* and *P. equestris* were named according to their relative homology with *A. thaliana* and *O. sativa* genes. Among them, the GAD subfamily was the largest, with 18 *PLP\_deC* genes, and the HDC subfamily was the smallest, with 6 members.

**Figure 1.** Phylogenetic analysis of *PLP\_deC* genes from *Dendrobium o*ffi*cinale*, *Phalaenopsis equestris*, *Oryza sativa*, and *Arabidopsis thaliana*. The maximum likelihood (ML) tree was created using IQ-TREE with 8 *D. o*ffi*cinale* (Do), 6 *P. equestris* (Pe), 15 *O. sativa* (Os) and 12 *A. thaliana* (At) PLP\_deC protein sequences. The red, green, and blue colors indicate GADs, HDCs, and AADs, respectively. Bootstrap supports are indicated at each branch.

To further analyze the gene structures and conserved motifs of the *PLP\_deC* family members in *D. o*ffi*cinale* and *P. equestris*, a total of 10 motifs were identified from the amino acid sequences of the *PLP\_deC* family members using the Multiple EM for Motif Elicitation (MEME) software (Figure 2). We checked these motifs to verify they are known domains by pfam and the motif logos were generated using online MEME program (Figure S1). Group I, which includes GAD sequences, harbors all the motifs; group II, which includes AAD sequences, harbors motifs 5, 6, 9, and 10; and group III, which includes HDC sequences, harbors motifs 4, 5, and 10. These results show that most *PLP\_deC* genes in the same subfamily have highly similar motifs, which supports their close evolutionary relationships and the reliability of the constructed phylogenetic tree. Remarkably, motif 10 and motif 5 are present in all subfamilies, but they are not part of the PLP\_deC domain. Thus, we speculate that they may perform other specific functions. In addition, we used the online Gene Structure Display Server to analyze the gene structures. The results showed that the number of exons in *PLP\_deC* genes ranged from 2 to 14. For example, there are 3–8 exons in subfamily GAD, 2–14 exons in subfamily AAD, and 5 or 7 exons in subfamily HDC. These results indicate that the number of exons in the *PLP\_deC* gene family has increased or decreased during evolution, providing a basis for functional differences among the homologous *PLP\_deC* genes (Figure S2).

**Figure 2.** Distribution of 10 putative conserved motifs in *PLP\_deC* proteins.
