*2.6. Analysis of the Expression Patterns of* PLP\_deC *Genes in* D. officinale

In plants, many stress responses are modulated or mediated by various signaling pathways that are inseparable from gene expression and regulation [21]. To investigate the responses of the *PLP\_deC* genes to different hormone treatments, we used qRT-PCR to analyze their expression under MeJA, ABA, and SA treatments.

In the ABA treatment, we found that *DoAAD1*, *DoAAD2*, *DoAAD3*, *DoGAD1*, *DoGAD3*, and *DoGAD4* reached their highest expression levels after 72 hours, and *DoGAD1* and *DoGAD3* were strongly upregulated (by more than 110-fold and 50-fold, respectively). The expression of two *PLP\_deC* genes (*DoGAD2* and *DoHDC1*) peaked at 96 h, and *DoGAD2* was strongly upregulated (more than 250-fold) (Figure 6). In the MeJA treatment, three *PLP\_deC* genes *(DoAAD3*, *DoGAD3*, and *DoHDC1)* showed strong upregulation at 2 h (Figure 7), while five PLP\_deC genes (*DoAAD1*, *DoAAD2*, *DoGAD1*, *DoGAD2*, and *DoGAD4*) were strongly upregulated after 4 h of treatment. In the SA treatment, the expression levels of *DoAAD1*, *DoAAD3*, *DoGAD1*, *DoGAD2*, and *DoHDC1* were strongly upregulated at 2 h (Figure 8). However, *DoAAD2* and *DoGAD3* were strongly upregulated after treated with SA for 72 h of treatment (over 645- and 508-fold, respectively).

**Figure 6.** The expression levels of *PLP\_deC* genes in *D. o*ffi*cinale* under abscisic acid (ABA) treatment. The x-axis represents the treatment time, and the y-axis represents the gene expression level. Error bars indicate the mean and standard deviation (SD). The asterisks indicate significant difference relative to the time 0. \*\* significant difference (*p* < 0.01), \* significant difference (*p* < 0.05).

**Figure 7.** The expression level of *PLP\_deC* genes in *D. o*ffi*cinale* under methyl jasmonate (MeJA) treatment stress. The x-axis represents the treatment time, and the y-axis represents the gene expression level. Error bars indicate the mean and standard deviation (SD). The asterisks indicate significant difference relative to the time 0. \*\* significant difference (*p* < 0.01), \* significant difference (*p* < 0.05).

**Figure 8.** The expression levels of *PLP\_deC* genes in *D. o*ffi*cinale* under salicylic acid (SA) treatment. The x-axis represents the treatment time, and the y-axis represents the gene expression level. Error bars indicate the mean and standard deviation (SD). The asterisks indicate significant difference relative to the time 0. \*\* significant difference (*p* < 0.01), \* significant difference (*p* < 0.05).

### **3. Discussion**

The type II PLP\_deC enzymes are an important group of carboxylases among the PLP-dependent enzymes. Many data indicate that PLP\_deCs show developmental, tissue-specific, and inducible transcript accumulation during plant development [19,22]. In this paper, we identified 8 and 6 *PLP\_deC* genes from the *D. o*ffi*cinale* and *P. equestris* genomes, respectively. According to the phylogenetic analysis, all the *PLP\_deC* genes from *A. thaliana*, *O. sativa*, *D. o*ffi*cinale*, and *P. equestris* were clustered into GAD, AAD, and HDC subclasses based on their high sequence similarity, which is consistent with the ML tree of *PLP\_deC* genes from the genomes of 18 species and previously published articles [17]. However, some of these genes might have evolved with different functions. For example, in tomato, *SlHDC19* and *SlHDC6* do not act on histidine but prefer tyrosine as their substrate [17]. Furthermore, many HDCs are biased toward serine rather than histidine based on biochemical analysis [18]. Therefore, although their sequences have high similarity, *PLP\_deC* genes have individual substrate specificities; we should perform an in-depth biochemical characterization to understand their precise functions [23].

Gene duplication is a common phenomenon in species and contributes to the generation of biodiversity during evolution [24]. To date, the chromosome assemblies of the *D. o*ffi*cinale* and *P. equestris* genomes have not yet been finished [25], and thus, the homologous genes of *D. o*ffi*cinale* and *P. equestris* cannot yet be clearly shown on the chromosomes. Therefore, we are unable to determine the type of replication events that have occurred between these species. To further understand the evolutionary patterns of the *PLP\_deC* genes, we calculated the Ka and Ks values of homologous gene pairs. We predicted that two gene pairs (*PeGAD1–DoGAD1* and *PeAAD2–AAD2*) are evolved from the genome-wide duplication events shared by *D. o*ffi*cinale* and *P. equestris*, because their values of Ks are 0.7 to 1.1 [26]. The Ka/Ks values in this experiment were less than 1 for all the homologous gene pairs except for *PeGAD2–PeGAD3*, implying that these gene pairs have undergone purifying selection during evolution. In addition, we noticed four homologous gene pairs (*PeGAD1–DoGAD1*, *PeAAD2–AAD2*, *PeAAD1–DoAAD3*, and *PeHDC1–DoHDC1*) had the comparatively high Ka/Ks values (>0.5), showing that these gene pairs have undergone rapid evolutionary diversification after duplication events in the course of evolution [24].

Analysis of *D. o*ffi*cinale PLP\_deC* gene expression in different tissues can help us better understand the tissue specificity of the *PLP\_deC* genes. Therefore, expression profiles for all the *PLP\_deC* genes were established using published RNA-sequence data. Among them, *DoAAD1*, *DoAAD2*, and *DoGAD3* were highly expressed in different tissues, indicating that these *PLP\_deC* genes play important roles during *D. o*ffi*cinale* growth and development. For example, GADs are involved in many cellular processes, including pollen-tube development in *Arabidopsis* and *Picea wilsonii* [25,27]. In this study, some cis-acting elements associated with particular tissues were identified in the *PLP\_deC* gene promoter regions, such as the O2-site required for seed expression and the CAT-box required for meristem organization. The corresponding *PLP\_deC* genes (such as *DoAAD1* and *DoAAD2*) might play important role in the formation of reproductive organs.

Many studies have suggested that the expression levels of *PLP\_deC* genes are also influenced by abiotic and biotic stresses [28–30]. Furthermore, plant hormones such as ABA, SA, and ethylene also modulate the expression of these genes [17,29,30]. In this study, we identified a number of cis-acting elements in the promoter regions of *PLP\_deC* genes in both *D. o*ffi*cinale* and *P. equestris*, such as MBS, MRB, Box 4, and ABRE. We found that these *PLP\_deC* genes contain at least one abiotic stress cis-element, which showed that they may contribute to biotic and abiotic stress responses. To further investigate the responses of the *PLP\_deC* genes to different hormones, we analyzed their expression with the treatments of MeJA, ABA, and SA by qRT-PCR. We observed that the *PLP\_deC* genes had significantly differential expression patterns under different treatments. Some of the *PLP\_deC* genes showed strong upregulation under the treatments, indicating that these genes play key roles in the abiotic stress responses of *D. o*ffi*cinale*. For example, *DoAAD2* was strongly upregulated (645-fold) after 72 h of SA treatment. Overall, we found that the *PLP\_deC* genes of *D. o*ffi*cinale* responded to abiotic

stress, such as MeJA, ABA, and SA stresses. These results provide strong evidence that the *PLP\_deC* genes in plants are involved in abiotic stress responses.

### **4. Materials and Methods**

#### *4.1. Materials and Treatments*

Seedlings of *D. o*ffi*cinale* were planted on Murashige and Skoog (MS) medium and placed in a tissue culture chamber at a constant temperature of 25 ◦C (16 h light/8 h dark) for 1 month. The tissue culture seedlings were then transferred to MS medium containing 1.0 mg/L 6-BA (biosharp, Shanghai, China), 0.1 mg/L NAA (Aladdin, Shanghai, China), and 30 g/L sucrose (Aladdin, Shanghai, China). The induced protocorms (PLBs) were supplemented with 1/2 MS liquid medium containing 0.1 mg/L α-naphthylacetic acid (NAA), 0.1 g/L whey protein hydrolysate, and 30 g/L sucrose (pH 5.8), and then cultured in darkness at 25 ◦C for 2 months. The PLBs were cut into 0.5 cm × 0.5 cm pellets, and 7 g of the pellet was inoculated into an Erlenmeyer flask containing 40 mL of MS medium. MeJA (100 μM methyl jasmonate; Aladdin, Shanghai, China), SA (100 μM salicylic acid; Aladdin, Shanghai, China), and ABA (100 μM abscisic acid; Aladdin, Shanghai, China) were filtered through 0.22 μm filter membrane and added to the MS medium, based on previously published articles [31]. After induction with SA and MeJA, samples were harvested at 0, 2, 4, 8, 24, 48, and 72 h. The original bulbs under ABA induction were harvested at 0, 24, 48, 72, 96, and 168 h. All samples were immediately stored at −80◦C after harvesting for RNA extraction. All results were based on three biological repeats and each biological repeat had three technical replicates. The extraction of total RNA from PLBs was carried out with Plant Total RNA Isolation Kit (Sangon Biotech, Shanghai, China) using 300 mg tissue homogenized in liquid nitrogen according to the manufacturer's protocol, which was subsequently reverse transcribed into the first DNA strand using a One Step RT-qPCR Kit (BBI Life Science, Shanghai, China).

#### *4.2. Screening and Identification of the* PLP\_deC *Genes*

We obtained the HMM (Hidden Markov Model) configuration file for the PLP\_deC domain (Pfam00282) from Pfam (http://pfam.xfam.org/). All *PLP\_deC* genes were then identified in the *D. o*ffi*cinale* and *P. equestris* genomes using HMMER software (*E*-value = 0.001) [32]. All candidate genes were submitted to Pfam and SMART for verifying the presence of the PLP\_deC domain. The sequences that did not contain the conserved domain and the redundant sequence of the repeat were removed, and finally, the *PLP\_deC* genes were obtained. In Table S1 are listed the sequences of the *PLP\_deC* genes of *O. sativa* and *A. thaliana* described in previous studies (add citations) and used in the present work [17].

### *4.3. Sequence Attribute Analysis and Phylogenetic Tree Construction of* PLP\_deC *Genes*

To analyze the sequence attributes and characteristics of the amino acids of the *PLP\_deC* family members, the isoelectric points (pIs) of the obtained PLP\_deC amino acid sequences were determined using online analysis with the ProtParam tool (https://web.expasy.org/protparam/) [33] and subcellular localization of 8 and 6 PLP\_deC-family protein sequences in *D. o*ffi*cinale* and *P. equestris* by Target P 1.1 (http://www.cbs.dtu.dk/ services / Target P). Properties such as molecular weight (MW) were predicted. The obtained PLP\_deC protein sequences were aligned using Clustal X [34], implemented in MEGA 5.0 [35], and a maximum likelihood (ML) phylogenetic tree was generated using IQ-TREE software [36] with 1000 bootstrap replicates. The PLP\_deC genes were classified according to the phylogenetic relationships. If two different species of genes are located in the phylogenetic tree at the same node and the sequence similarity is more than 80%, we consider two of these are homologous genes [37]. The conserved motifs on the orchid sequences of PLP\_deC were defined by MEME (http://meme-suite.org/) using the following parameters: maximum number of motifs = 10, number of repetitions—any, and only motifs with an *E*-value < 0.01 were retained for further analysis. The motif

logos of the PLP\_deC domains were generated using online MEME program (Figure S2) [38], and GSDS was used to determine the exon–intron structure (http://gsds.cbi.pku.edu.cn /) [39].
