*3.3. Biological Mechanism of Verbena in Response to Drought Stress*

The protective mechanisms of plants in response to stress are regulated by alterations in the expression level of stress-responsive genes. Among the DEGs assigned to KOG and COG, genes related to plants' stress response accounted for quite a high proportion; 8.50% and 11.00%, respectively. We performed GO and KEGG pathway enrichment analysis and excavated a group of important drought-responsive genes related to multiple biological mechanisms of plant energy production,

hormone synthesis, cell signaling, and metabolism. In this study, there are 12, 25 and 24 genes differentially expressed in "glyoxylate and dicarboxylate metabolism", "citrate cycle (TCA cycle)" and "glycolysis/gluconeogenesis" pathway, respectively, and all of them were up-regulated. This indicates that the energy production of Verbena changed a lot, and the main method of providing ATP was changed from the photosynthetic phosphorylation to the oxidative phosphorylation.

ABA is one of the most important factors in abiotic stress response, and is involved in almost all plant activities, such as photosynthesis, ionic homeostasis, and antioxidant defense [43]. The quantities of gene expression controlling the key enzymes, such as PYL and PP2C in the ABA signaling pathway, have changed a lot. The vicissitudinous DEGs of PYL activate the high expression of PP2C and furthermore promote stomatal closure and decrease transpiration to reduce water loss. Under the "plant hormone signal transduction" pathway, in addition to the enzymes of ABA, the level of genes that control PR1 in the "salicylic acid (SA)", JAZ and MYC2 in the "jasmonic acid (JA)", GH3 and SAUR in the "auxin", and MPK6 together with EBF1\_2 in "Ethylene" pathways, were almost all up-regulated (Table 2). This is in line with previous findings that the changes of hormonal synthesis and signal transduction are the conservative mechanisms by which plants response to adverse circumstances. It has been demonstrated that NO (nitric oxide) can act as a signaling molecule to activate ROS-scavenging enzymes in drought stress [44]. Under the pathway of "Nitrogen metabolism", we observed that the gene expression levels of NR and NirA in the assimilatory nitrate reduction pathway were down-regulated, and the expression of GLT1 and GLUL in the glutamate synthase pathway were up-regulated (Table 6). The glutamate synthase pathway leads to the process of glutamate metabolism and the production of ammonia, and a sufficient amount of oxidized glutamic acid will ensure that there is sufficient carbon skeleton for the tricarboxylic acid cycle to function effectively [45]. Therefore, we can speculate that improving the level of nitrogen metabolism is a very efficient method to help plants resist abiotic stress.

Carbohydrate metabolism is the center of the entire biological metabolism and involves the protein metabolism, lipid metabolism, nucleic acid metabolism and secondary metabolites production. Studies have shown that in rice [46], sugar can be used as a signal to induce the expression of genes associated with abiotic stress. Garg A.K. et al. [47] found that the accumulation of trehalose in rice enabled the transgenic rice to exhibit high salt tolerance, drought tolerance, and low-temperature stress. In this study, there were 28 and 12 DEGs in "Starch and sucrose metabolism" and "Pentose phosphate pathway", respectively, most of which were up-regulated, which indicates the importance of sugar for Verbena to cope with drought stress. Furthermore, we found a series of pathways for the up-regulation of DEGs expression: protein metabolism such as "Biosynthesis of amino acids", lipid metabolism such as "Fatty acid metabolism" and "Glycerophospholipid metabolism", nucleic acid metabolism such as "Pyrimidine metabolism" and "Purine metabolism", and metabolism of secondary products such as "alpha-Linolenic acid metabolism" and "Flavone and flavonol biosynthesis". All of these show that carbohydrate metabolism is very important for Verbena to improve its tolerance to adverse environmental conditions.

Moreover, "ubiquitin mediated proteolysis", an energy-consuming, highly efficient and highly directional protein degradation process, is also noteworthy. It plays an important role in many aspects, such as modulation of the immune and inflammatory responses, the regulation of cell cycle, control of signal transduction pathways, development and differentiation etc. [48,49]. In the present study, there are 16 DEGs in this pathway (Table 7), all of which up-regulate. Half are involved in the regulation of ubiquitin conjugating enzyme, and the others are involved in the regulation of ubiquitin ligase. At present, the understanding of this process is still very limited. The genes participated in this process during drought stress, and the mechanism of the enzymes which were regulated by these genes has yet to be studied.


**Table 6.** DEGs in "Nitrogen metabolism" pathway of KEGG.

**Table 7.** DEGs in "Ubiquitin mediated proteolysis" pathway of KEGG.


We found evidence that Verbena responds to drought stress by altering energy synthesis pathways, decreasing transpiration, resetting hormone secretion levels, and increasing cell osmotic pressure and glucose metabolism. In general, Verbena's defensive response, like most plants under stress conditions, is a process of rebuilding physiologically, biochemically and metabolically, from the growth-oriented to the defensively based one. The genes mentioned in Section 3 are listed in Tables 1–7 and after counting the TFs to which these genes belong, we constructed the MYB TF phylogenetic tree using the reported abiotic stress genes to prepare for the next work (Figure SA8).
