FIGURE 11 Principal component analysis of secondary metabolites of A. trifoliata in different habitats
4  | DISCUSSION
Environmental stress can not only inhibit plant growth, but also stimulate the synthesis and accumulation of plant secondary metabolites (Zhang et al., 2020). However, the biggest characteristic of rocky desertification habitat is that it does not keep water, resulting in a large amount of water loss. Thus causing certain drought stress to plants. The biomass of various organs of A. trifoliatasignificantly decreased in the rocky desertification habitat, which indicates that the desertification habitat has caused a certain abiotic stress on A. trifoliata , and the metabolic physiological processes of its plants will inevitably change.
4.1  | Effects of rocky desertification on the contents of basic secondary metabolites in A. trifoliata
The secondary metabolites such as phenols and flavonoids are closely related to plant stress defense (Sanchez et al., 2011). In this study, we found that rocky desertification could promote the increase of total phenol content in roots, stem and leaf of A. trifoliata , which was the same as that of total phenol secretion of Desi ajwain (Trachyspermum ammi L.) under drought stress (Azhar et al., 2011). The possible reason is that the increase of total phenol content can help to reduce the water potential in cells, prevent the loss of water in cells; At the same time, phenol can also remove reactive oxygen species produced under drought (Nakabayashi et al., 2015). In this study, it was found that the content of polyphenols in leaves changed the most, which may be due to the fact that leaves are the largest organ of plants exposed to the outside world, and they are most seriously exposed to oxidative stress. Therefore, they secrete a large number of polyphenols, remove reactive oxygen species and free radicals through hydrogen atom transfer mechanism, and are used for the synthesis of antioxidants to resist oxidative damage (Ping et al., 2017).
Tannins and flavonoids belong to the phenolic compounds of plant secondary metabolites, which are good free radical scavengers and lipid peroxidation inhibitors (Dicko et al., 2005). The results showed that the content of tannin in A. trifoliata increased rapidly in the early stage and decreased slowly in the later stage. The possible reason is that water shortage in rocky desertification habitat can induce the increase of secondary metabolism related enzyme activities in A. trifoliata , so a large number of secondary metabolites are activated. Finally, it is transported to various organelles to repair the damage of membrane system and improve the tolerance of plants to drought stress, which is a self-protection mechanism for plants to cope with stress (Castellarin et al., 2007). The tannin content would slow down and decrease in the later rocky desertification habitat. The possible reason is that with the aggravation of stress, the plant organ damage is serious, the metabolism speed in the body slows down, resulting in the reduction of carbon sources required for the synthesis of phenols. Therefore, the tannin content in the plant does not increase or even decrease. Flavonoids usually have special structures such as hydroxyl, double carbon bond, glycosylation, acylation, methylation, etc., which can effectively resist oxidative damage induced by stress. At the same time, they can also complete antioxidant function by preventing the generation of reactive oxygen species and scavenging reactive oxygen species (Agati et al., 2010; Singanusong et al., 2015). In this study, we found that rocky desertification habitat can improve the content of flavonoids in the root and stem of A. trifoliata , which is the same as Zahir et al., (2014) study on water stress in Silybum marianum. The possible reason is the water deficiency caused by rocky desertification, which affects the up regulation of genes related to flavonoid metabolism pathway, such as MYB transcription factor, flavone-3-hydroxylase (F3H) and Flavonol Synthase (FLS) (Ying et al., 2018; Fan et al., 2018), thus increasing the content of flavonoids. However, there is no obvious rule for the change of flavonoids content in A. trifoliata leaves in rocky desertification habitat in the two bases. The possible reason is that the flavonoids content in leaves is less affected by rocky desertification habitat, so there is no obvious change rule.
4.2  | Effects of rocky desertification on enzyme activities of phenylpropanoid pathway in A. trifoliata
The changes of biological and abiotic factors do not directly affect the synthesis of plant secondary metabolites, but affect the synthesis of secondary metabolites by affecting the expression of key genes and key enzyme activities in secondary metabolism (Havaux and Kloppstech 2001). PAL, C4H and 4CL are key enzymes in phenylalanine metabolism pathway, and their activities are affected by environmental factors such as light, temperature and water (Dupont and Aksnes 2010). This study found that A. trifoliata in rocky desertification habitat can improve the activities of pal, C4H and 4CL in roots, 4CL in stems and PAL, 4CL in leaves. Obviously, different enzymes have different responses to rocky desertification habitat, and the changes in different tissues and organs are also different. In rocky desertification habitat, the three enzymes in roots were significantly increased, which may be due to the root as the main organ of plant drought resistance, its flavonoids content is also the most, so its response changes are also the most obvious, especially pal and C4H changes are the most obvious. It can be seen that the appropriate degree of drought stress can promote the accumulation of flavonoids by stimulating the expression and activity of key enzymes involved in flavonoid biosynthesis. This is consistent with the research results of Cheng et al., (2018) that moderate drought stress can promote baicalin accumulation by stimulating the key enzyme activity and expression of Baicalin biosynthesis.
4.3  | Effects of rocky desertification on oleanolic acid and α-hederagenin contents in A. trifoliata
Oleanolic acid and α-hederagenin are the two most important medicinal components of A. trifoliata . They are triterpenoids in terpenoids. They can not only participate in interspecific competition as interspecific sensing compounds (Arimura et al., 2000), but also be used as raw materials for spices, flavorings and cosmetics (Martin et al., 2003), pesticides and industrial raw materials (George et al., 2015), As well as anti-tumor, anti-inflammatory, antibacterial, antiviral, antimalarial, promoting percutaneous absorption, prevention and treatment of cardiovascular disease, hypoglycemic and other biological activities (Chen et al., 2021; Sun et al., 2019). However, their ecological functions, biosynthetic pathways and environmental factors are not very clear. In this study, we found that rocky desertification could increase the contents of oleanolic acid and α-hederagenins in the roots, stems and leaves of A. trifoliata . The possible reason is the water stress caused by rocky desertification habitat, which induces the expression of BPW and BPY genes and reduces the expression of BPX genes, resulting in the accumulation of triterpenoid saponins (Yin et al., 2015). This is the same as the result of Chen et al., (2011) which found that moderate drought would increase oleanolic acid in Prunella vulgaris L., and the possible reason is that oleanolic acid has antioxidant effect, which can help A. trifoliata resist the oxidative damage caused by rocky desertification habitat (Zhao et al., 2013). However, the role of α-hederagenins under drought stress is still unclear, so it needs to be studied. At the same time, this study found that rocky desertification habitat did not affect the distribution of oleanolic acid content in all organs of A. trifoliata , which was still root > stem > leaf. However, in Huaxi base, the peak period of oleanolic acid content in roots was delayed to a certain extent, but not in Guanling base. The possible reason is that A. trifoliata adapts to rocky desertification habitat stress for a long time in Huaxi base, which causes the peak period of oleanolic acid content to shift. However, the content peak and distribution of α-hederagenins in various organs did not change.
Although the biomass of roots, stems and leaves of A. trifoliatawill decrease in the rocky desertification habitat, the content of medicinal ingredients has increased significantly. This not only brings new economic benefits to A. trifoliata for restoring rocky desertification habitats, but also further improves the quality ofA. trifoliata medicinal ingredients.
5  | CONCLUSION
The response of secondary metabolites of A. trifoliata to rocky desertification was studied. The results showed that the rocky desertification habitat reduced the biomass of A. trifoliata , but significantly increased the tannin content in the leaves, the flavonoid content in the roots, and the total phenol content in the roots, stems and leaves, and reduced the flavonoid content in the leaves; The activities of PAL, C4H and 4CL in roots, 4CL in stems and PAL, 4CL in leaves of A. trifoliata in rocky desertification habitat were increased. The content of oleanolic acid and α-hederagenin in the root, stem and leaf of A. trifoliata was increased in rocky desertification habitat. In addition, the content of oleanolic acid in the root of A. trifoliata was the highest from July to August, which was suitable for picking medicine. The content of α-hederagenin in the stem was the highest in July, which was suitable for picking medicine. The principal component analysis showed that the main response index of secondary metabolites of A. trifoliata in rocky desertification habitat was total phenol. The results of this study are of great signifificance to cultivators and researchers of medicinal plants.