Glycyrrhiza uralensis Fisch. (G. uralensis), used as a common Chinese medicinal herb, contains abundant pharmacological active substances, which have attracted more attention (Hosseinzadeh and Nassiri-Asl, 2016). In the desert, although often suffering from drought and salt stresses, it is still an important sand fixation and windbreak plant (Zhou et al., 2020). In recent years, the wild resources are sharply declining and turning endangered, so the market demand has been markedly increasing for the cultivated G. uralensis (Chen et al., 2017). Therefore, improving the quality, yield, and stress tolerance of G. uralensis cultivated is the key task.

The glycyrrhizic acid and liquiritin biosynthesis pathway of G. uralensis is well known. In this pathway, HMGR catalyzes HMG-CoA and NADPH into MVA first, and farnesyl diphosphate (FDP), squalene synthase enzyme (SQS), and squalene epoxidase (SE) were involved in the subsequent reaction (Yan et al., 2014; Zheng et al., 2014). Then, β-amyrin synthase (β-AS), lupeol synthase (LUS), and cycloartenol synthase (CAS) are responsible for the synthesis of triterpene saponins, betulinic acid, and phytosterols, respectively (Mochida et al., 2017). Specifically, cytochromes P450s, UGAT, and CHS play crucial roles in the final step of major medicinal ingredient biosynthesis in G. uralensis (Xu et al., 2016; Chen et al., 2017). The content of compounds and the expression level of key genes have significantly changed in glycyrrhizic acid and liquiritin biosynthesis pathway during abiotic stress, salt stress specifically caught our attention (Wang et al., 2015, 2017; Zhou et al., 2017; Yeo et al., 2018; Kaur and Ganjewala, 2019). Shirazi et al. (2019) reported that salt stress significantly upregulated the expression level of SQS, β-AS, CAS, LUS, and some CYPs genes and increased the content of glycyrrhizin in Glycyrrhiza glabra, which illustrated that salt stress inhibited in the glycyrrhizin biosynthesis pathway. In conclusion, we provided a basis for further studies concerned with the relation between salt stress and glycyrrhizic acid pathway and liquiritin pathway in G. uralensis.

Endophytes colonized the internal tissues of host plants, promoted growth conditions, improved stress tolerance, and so on, so it is regarded as a low-cost and eco-friendly technology to enhance crop productivity under salt stress (Vaishnav et al., 2019). In promoting the growth of plant aspect, Mesorhizobium sp. and Pseudomonas extremorientalis endophytes could improve yield, nodule numbers, and nitrogen contents of shoot and root in salt-stressed G. uralensis (Egamberdieva et al., 2016). In promoting compound biosynthesis of plant aspect, endophyte Methylobacterium oryzae regulated ethylene metabolism in salt-stressed Oryza sativa by improving H⁺ ATPase activity and decreasing 1-Aminocylopropane-1-carboxylic acid (ACC) accumulation and ACC oxidase activity (Chatterjee et al., 2019). Also, endophytes increased withanolide A content in Withania somnifera, and significantly upregulated the expression level of genes in withanolide and sterol biosynthetic pathways including HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1, and CYP710A1 (Kushwaha et al., 2019). In conclusion, endophytes can effectively mitigate high-salinity stress and improve the stress tolerance of plants, but the effect of Bacillus cereus on G. uralensis is still not fully reported, particularly in the glycyrrhizic acid and liquiritin biosynthesis pathway aspect.

Consequently, in this study, we hypothesized that B. cereus can promote the growth of seedlings and increase the contents of medicinal ingredients (liquiritin, liquiritigenin, isoliquiritigenin, glycyrrhizic acid, and glycyrrhetinic acid) of G. uralensis under control and salt stress conditions. Furthermore, we want to explain the effect that B. cereus increased the contents of medicinal ingredients under different conditions based on the molecular level. Finally, we expected to unravel the underlying effect for the interactions of B. cereus and salt stress, provide an efficient solution for promoting the quality of G. uralensis cultivation, and set a solid foundation for B. cereus promoting the biosynthesis of glycyrrhizic acid and liquiritin.

In general, salt is a necessary nutrient element for the normal growth of plants and also plays an important role in the physiological and biochemical functions of plants (such as osmotic adjustment, antioxidant system, and carbon and nitrogen metabolism), but excessive concentration will cause plant death (Jose et al., 2017; Kanagaraj and Sathish, 2017). Salt stress inhibited the root growth of plants, reduced the activity of root tip cells mitotic, and increased abnormalities such as chromosome breaks and bridges and formation of micronuclei, which belongs to the cytotoxic effect (Kiełkowska, 2017). In this experiment, salt stress significantly decreased the root length of seedlings. Salt stress would affect the absorption of water and nutrients by damaging the roots of plants and further affect the supply of water and nutrients above the ground (Guo et al., 2015). Surprisingly, one biological method is the application of plant growth-promoting bacteria to decrease the deleterious effect of salinity (Trdan et al., 2019). Under salt stress, B. cereus significantly increased the root length and the number of lateral roots, but have no significant effect on dry weight, which could attribute to need more time for accumulate the effect of inoculation B. cereus on dry weigh. Plant growth-promoting bacteria that can alleviate salt stress have been reported in pepper (Amor and Cuadra-Crespo, 2012), barley (Suarez et al., 2015), okra (Habib et al., 2016), and Medicago sativa L. (Ansari et al., 2019). In addition, we also found that the effect of B. cereus on regulating the growth condition was greater under salt stress than these under the control condition, probably because the microorganisms can secrete ACC deaminase, degrade ethylene synthesis precursors, and regulate ethylene concentration under salt stress, which is an important mechanism for microorganisms to enhance plant stress resistance (Pande et al., 2016). In summary, B. cereus can promote the growth of G. uralensis seedlings, but the effect was greater under salt stress than that under the control condition.

In the past, Ponce et al. (2004) observed inoculation of Glomus intraradices increased flavonoid content from roots and shoots of Trifolium repens through increasing the root colonization, which may refer to the plant metabolism to secrete different flavone derivatives. At present, it is difficult to explain meticulously the molecular mechanism of endophytes on active compound biosynthesis because it is difficult to evaluate the potential functional relationships in traditional Chinese medicine, so we will try to analyze the molecular effect of B. cereus on improving the content of active compounds in G. uralensis based on transcriptome and PCR results. First, we observed that B. cereus increased glycyrrhizin content under the control condition and the contents of glycyrrhizic acid and glycyrrhetinic acid under salt stress (Figure 2). Second, our KEGG annotation results revealed that B. cereus increased liquiritigenin content is closely related to regulate flavone and flavonol biosynthesis under the control condition (Figure 4A; Berim and Gang, 2016), and B. cereus increased contents of glycyrrhizic acid and glycyrrhetinic acid are closely related to the regulation of phenylpropanoid biosynthesis under salt stress (Figure 4B; Li et al., 2016).

Liquiritigenin is one of the major active metabolites in G. uralensis, which belongs to flavonoid compounds and plays important physiological roles in protecting plants from biotic and abiotic stresses (Hosseinzadeh and Nassiri-Asl, 2016). Since liquiritigenin belongs to flavonoid compounds, our results explained that B. cereus increased the liquiritigenin content by upregulating the expression level of genes on the flavone and flavonol biosynthesis pathway under the control condition. Under the salt stress condition, B. cereus increased the contents of glycyrrhizic acid and glycyrrhetinic acid, this is a complicated process. These results are also consistent with our correlation analysis and PC analysis results. According to previous studies, glycyrrhizic acid and glycyrrhetinic acid are synthesized mainly by the MVA pathway, and this pathway is closely related to the cytochrome P450s (Pandian et al., 2020). Cytochrome P450s may account for the biosynthesis of triterpene saponins, phenylpropanoid, and fatty acids in plant-specific organs and further modified oxidation and glycosylation (Yendo et al., 2010; Liao et al., 2020). Phenylpropanoid compounds play a pivotal role particularly in signaling molecules both within the plant and in communications with other microbes, response to environmental conditions, and improving crop yield and quality (Ali and McNear, 2014). Also, we found that more than sixteen cytochrome P450s have been implicated in the phenylpropanoid metabolism pathway because most are derived from phenylalanine by the phenylpropanoid pathway leading to 4-coumaroyl-CoA (Pandian et al., 2020). In our study, B. cereus increased the contents of glycyrrhizic acid and glycyrrhetinic acid, upregulated the expression level of CYP88D6 and CYP72A154 genes, and upregulated the level of DEGs in phenylpropanoid biosynthesis under salt stress, which showed that glycyrrhizic acid and glycyrrhetinic acid, CYPs, and phenylpropanoid biosynthesis are closely related. Moreover, the complex relationships between B. cereus and glycyrrhizic acid and liquiritin biosynthesis pathway remain to be further studied.

In contrast, we interpreted the effect of B. cereus improving the contents of active compounds of G. uralensis based on PCR results. According to a large number of reports, we summarized the major active compounds and the enzymes of the secondary metabolic biosynthesis pathway in G. uralensis (Figure 10). In past studies, the effect of high salt concentration on the content of effective ingredients depends on different plant types, growth stages, and action sites (Wang et al., 2016). Behdad et al. (2020) found that 100 mM NaCl significantly upregulated the expression level of key genes (β-AS, CYP88D6, and CYP72A154) in Glycyrrhiza glabra L. involved in triterpenoid saponin biosynthesis and directly increased the content of glycyrrhizic acid. Shirazi et al. (2019) also found that salt stress upregulated the expression level of key genes (including SQS, β-AS, LUS, CAS, CYP88D6, and CYP93E6) in Iranian licorice. Moreover, Wang et al. (2020) reported that 200 mM NaCl upregulated the expression level of key enzymes (β-AS, CYP88D6, and CYP72A154) involved in the glycyrrhizic acid biosynthesis pathway in G. uralensis. Our results are different from theirs, and in this study, salt stress significantly downregulated the expression level of HMGR, β-AS, and UGAT genes, upregulated the expression level of CYP5 and CHS genes, significantly decreased glycyrrhetinic acid content, and significantly increased the contents of liquiritin and liquiritigenin, which is consistent with the biosynthesis pathway of active compounds in G. uralensis. Salt stress downregulated the expression level of HMGR, β-AS, and UGAT genes, reducing the content of compounds in glycyrrhizic acid synthesis pathway; at the same time, salt stress upregulated the expression level of CYP5 and CHS genes, increasing the content of compounds involved in the liquiritin synthesis pathway.

A large number of studies have found that endophytes can improve the accumulation of active compounds in plants. Native endophytes of Coleus forskohlii significantly promoted the plant growth (shoot weight, oil content, and oil yield) and increased the contents of davanone and ethyl cinnamate in Andrographis paniculata, Pelargonium graveolens, and Artemisia pallens (Mastan et al., 2019). Endophytes Phoma sp., Alternaria sp., Bipolaris sp., and Cladosporium sp. significantly increased seed germination, promoted plant growth, and increased the contents of flavonoid and phenolic in rice (Khan et al., 2017). Particularly in the salt stress, it has been reported that endophytes have many beneficial effects on salt-stressed plants including the production of scavengers, ACC deaminase enzyme, nitrogen fixation, production of compatible solutes, antibiotics, and phytohormones (Fouda et al., 2018). Amanifar et al. (2019) found that arbuscular mycorrhizal increased the content of glycyrrhizic acid and upregulated the expression level of β-AS and P450 genes in Glycyrrhiza glabra under salt stress. This is similar to our research results that B. cereus upregulated the expression level of key genes and the content of active compounds involved in the glycyrrhizic acid and liquiritin biosynthesis pathway, which indicated that B. cereus induced the accumulation of major active compounds in G. uralensis through the upregulation expression level of MVA and downstream pathway genes such as HMGR, β-AS, SQS, CHS, LUS, UGAT, CYP72A154, CYP88D6, and SE. Moreover, the regulatory effect of B. cereus was different under control and salt stress conditions. HMGR acts as the catalyst for the first committed reaction of the mevalonate pathway, which is critical for plant adaptations against harsh environmental conditions (Zheng et al., 2021), so the expression level of HMGR gene was higher by B. cereus under salt stress. Moreover, B. cereus upregulated the expression level of many key genes of G. uralensis in both control and salt stress conditions, but only increased the content of liquiritigenin under the control condition and increased the contents of glycyrrhizic acid and glycyrrhetinic acid under the salt stress condition. Interestingly, this result is consistent with the results of transcriptome analysis, which explained that the effect of B. cereus on the glycyrrhizic acid and liquiritin biosynthesis pathway of G. uralensis under control and salt stress conditions was different.

In this study, B. cereus improved the growth condition and the contents of major medicinal components of G. uralensis seedlings. Interestingly, the effect of B. cereus on the seedling growth and the glycyrrhizic acid and liquiritin biosynthesis pathway was different under control and salt stress conditions. In promoting the seedling growth aspect, the effect of B. cereus under the salt stress condition was greater than that under the control condition. Also, the effect of B. cereus on the accumulation of active compounds was different under control and salt stress conditions based on transcriptome and PCR results. B. cereus increased liquiritigenin content under the control condition, which is closely related to regulate flavone and flavonol biosynthesis, and increased the contents of glycyrrhizic acid and glycyrrhetinic acid involved in the phenylpropanoid biosynthesis pathway under the salt stress condition. Moreover, our study provided some theoretical basis for improving the contents of major active compounds and enhancing salt tolerance of G. uralensis and the development and utilization of B. cereus and molecular breeding of G. uralensis, which will help the further study in G. uralensis.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, GSE187003.

YZ and XZ contributed to the experiment design. WZ and YZ performed the material preparation and data collection. DL and YZ analyzed the data. YZ wrote the first draft of the manuscript. All authors commented on previous versions of the manuscript, read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.