*3.2. The Enrichment and Pathway Analysis of DEGs in GO and KEGG Databases*

*3.2. The Enrichment and Pathway Analysis of DEGs in GO and KEGG Databases* The chlorophyll content decreased sharply in the early stage of drought stress, and eventually increased to a lower level than T01. The 16 DEGs related to "porphyrin and chlorophyll metabolism" are listed in Table 1, and the enzymes of porphyrin metabolism were almost all up-regulated, while the enzymes of chlorophyll metabolism were almost all down-regulated. Analyzing the genes involved in the regulation of stomatal closure by ABA (abscisic acid), we found that some of the genes' The chlorophyll content decreased sharply in the early stage of drought stress, and eventually increased to a lower level than T01. The 16 DEGs related to "porphyrin and chlorophyll metabolism" are listed in Table 1, and the enzymes of porphyrin metabolism were almost all up-regulated, while the enzymes of chlorophyll metabolism were almost all down-regulated. Analyzing the genes involved in the regulation of stomatal closure by ABA (abscisic acid), we found that some of the genes' expression levels of PYL and PP2C were significantly up-regulated (Table 2), and according to the analysis of the "carbon fixation in

expression levels of PYL and PP2C were significantly up-regulated (Table 2), and according to the

photosynthetic organisms" pathway, we found that a number of genes related to "C4-Dicarboxylic acid cycle and carbon fixation pathways in prokaryotes" process showed an upward trend. This indicates that under drought stress, the photosynthesis of Verbena may be mainly inhibited by stomatal closure, and at the same time it may provide an adequate carbon source for photosynthesis by enhancing the biological carbon sequestration pathway. The DEGs in "photosystem II" and "photosystem I" of CC also showed a high degree of enrichment, indicating that drought had a great impact on the photosynthesis of Verbena, which is the direction we should focus on. In MF, "acyl-CoA dehydrogenase" and "oxidoreductase" are obviously enriched. Previous study has shown that deficiencies of acyl-CoA dehydrogenases can lead to disorders of fatty acid oxidation, leading to life-threatening metabolic disorders [30]. It is known that drought stress will produce a large amount of reactive oxygen species (ROS) in plants and start the massive production of oxidoreductase such as SOD. Therefore, the increase of "acyl-CoA dehydrogenase" and "oxidoreductase" is of great significance for plants in responding to drought stress.


**Table 1.** DEGs (analysis of differentially expressed genes) in "Porphyrin and chlorophyll metabolism" pathway of KEGG (Kyoto Encyclopedia of Genes and Genomes).

**Table 2.** DEGs related to hormone synthesis in response to drought stress.


In the KEGG database, the first three pathways with the highest DEGs enrichment factors are "photosynthesis antenna proteins", "betalain biosynthesis" and "flavone and flavonol biosynthesis". After comparison, the homologous genes with almost all the down-regulated DEGs (Table 3) in "photosynthesis antenna proteins" pathway are related to LHC proteins, which may be involved in the drought resistance of plants and play an important role in crop environmental adaptability and yield [31]. Alberte R.S. et al. [32] showed that LHC proteins are a target easily attacked by water stress, and the loss of chlorophyll, the increase of chlorophyll a/b ratio and the decrease of photosynthetic unit under water stress are all caused by the decrease of LHC proteins. Vappaavuovi E. et al. [33] also confirmed that water stress reduced the LHC protein complex. In addition, some studies have showed that LHC proteins may be involved in the partial regulation of ABA signaling and play an active role in guarding cell signaling—these proteins may be induced by ABA and positive regulates ABA to inhibit stomatal opening [34,35]. Therefore, the decrease of LHC proteins will inhibit the production of ROS. However, due to the insufficiency research of LHC proteins, further research on its response to abiotic stress is needed. "Betalain biosynthesis" pathway has three distinctly up-regulated genes (Table 4). Betaine is the trimethyl derivative of the amino acid glycine, an efficient methyl donor that promotes fat metabolism and protein synthesis and the increasing of betaine biosynthesis has been shown to play an important role in osmoregulation of plants, which can help plants to withstand drought stress [36]. The "Flavone and flavonol biosynthesis" pathway has four distinctly up-regulated genes with an enrichment factor of 4.71, and studies have shown that the flavonoid substance may contribute to antioxidant functions in response to drought stress [37]. At present, there is still a lack of research on the changes in the content of secondary metabolites that have economic or medicinal value in Verbena under abiotic stress, but according to the schematic pathway figures of betalain and flavone (Figure 9a,b), we can preliminarily speculate that the content of flavonoids and betaine were up-regulated under drought conditions, while other plants also have similar research conclusions. Xing W. et al. [38] have shown that under drought stress, the endogenous leaf glycine betaine level of *A. thaliana* L. increased about 18-fold over that in the control plants, and similar results were also found in Pyrus bretschneideri Rehd [39] and *Hordeum vulgare* L. [40]. From the pathway of "flavone and flavonol biosynthese", we can see that all DEGs are up-regulated (Table 5) and there are two kinds of useful downstream production—luteolin and quercetin. Studies have shown that drought increased the accumulation of luteolin in *Ligustrum lucidum* Ait. [37], and quercetin also showed a positive impact on *Vigna unguiculata* L. Walp. [41], but in *Cabernet Sauvignon* [42], quercetin showed no obvious change under water stress. Therefore, further experiments are needed to investigate the effects of abiotic stress on secondary metabolite content in Verbena.


**Table 3.** DEGs in "Photosynthesis-antenna Proteins" pathway of KEGG.



*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 12 of 20

**Table 4.** DEGs in "Betalain biosynthesis" pathway of KEGG.


(**b**)

**Figure 9.** Two schematic pathway figures of "Betalain biosynthesis" and "Flavone and flavonol biosynthesis": (**a**) Effect of drought stress on the expression of genes associated with betaine. (**b**) Luteolin and quercetin metabolism. **Figure 9.** Two schematic pathway figures of "Betalain biosynthesis" and "Flavone and flavonol biosynthesis": (**a**) Effect of drought stress on the expression of genes associated with betaine. (**b**) Luteolin and quercetin metabolism.
