Differential Expression of Calycosin-7-O-β-D-glucoside Biosynthesis Genes and Accumulation of Related Metabolites in Different Organs of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao Under Drought Stress

Calycosin-7-O-β-D-glycoside (CG), as a flavonoid, plays an important role in the abiotic stress response of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao (A. mongholicus). CG is also an active ingredient in A. mongholicus with high medicinal value. However, the response mechanism of the CG biosynthetic pathway of drought stress is not clear. In this research, drought stress was inflicted upon A. mongholicus, and the variations in flavonoid metabolites and the correlating gene expression in CG biosynthesis were studied in roots, stems, and leaves of A. mongholicus by UHPLC-MRM-MS/MS and qRT-PCR. Drought stress reduced the dry weight and increased the content of malondialdehyde (MDA) and proline. Drought was beneficial to the accumulation of L-phenylalanine and 4-coumaric acid in leaves and promoted the accumulation of all target compounds in the roots, except calycosin. Overexpression of AmIOMT was observed in the leaves, but the content of formononetin which is the product of isoflavone O-methyltransferase (IOMT) catalysis was higher in stems than in leaves. This research aims to further understand the acclimation of abiotic stress and the regulation mechanism of flavonoid accumulation in A. mongholicus.


Introduction
Secondary metabolites are an important group of compounds essential for plant acclimation and survival in varying environmental conditions, which can be classified into three major types, including terpenoids, nitrogen-containing metabolites, and phenolics [1]. As antioxidants, phenolic compounds (tannins, flavonoids, and lignin) can inhibit the generation of Region, Northwest China. Under a natural photoperiod (approximately 14/10 h light/dark cycle) and 26 ± 4℃/16 ± 3℃ day/night cycle from March to June 2018. Seeds of A. mongholicus were purchased from Inner Mongolia Tianchuang Pharmaceutical Technology Co., Ltd. The seeds were immersed in water at 100 °C for 90 s, followed by soaking in water at 30 °C for 3 h, which were then sown in plastic pots (17 cm in diameter and 25 cm in height), 40 per pot on March 15, 2018. The soil organic matter content is 12.92 ± 1.06 g/kg, total nitrogen is 0.86 ± 0.07 g/kg, alkali nitrogen is 53.67 ± 10.69 mg/kg, total phosphorus is 0.36 ± 0.07 g/kg, available phosphorus is 43.77 ± 0.93 mg/kg, total potassium is 10.77 ± 0.87 g/kg, available potassium is 24.25 ± 3.55 mg/kg, and pH = 8.14 ± 0.03. Water is poured once every 4-5 days after the seeds are germinated.
When the plant height of most seedlings was about 15 cm, seedlings with roughly equal height were selected and randomly divided into two groups. One group was watered well (control group), and the other group was not watered to simulate 15 days of drought stress and then rewatered for 7 days. On days 0, 3,6,9,12,15, and 21, six pots were randomly selected from each group. Root and shoot of the seedlings were separated, which were then washed with distilled water, gently blotted dry with a filter paper, weighed for fresh weight  [16]. PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl CoA ligase; CHS, chalcone synthase; CHR, chalcone reductase; CHI, chalcone isomerase; IFS, isoflavone synthase; IOMT, isoflavone O-methyltransferase; I3′H, isoflavone3′-hydroxylase; UCGT, UDP-glucose: calycosin-7-O-glucosyltransferase (FW), and then were oven dried at 60℃ for 48 h for dry weight (DW) determination, respectively. Samples of different organs (roots, stems, leaves) collected on the 15th day and 21st day were used for RNA and compound extraction.
Soil samples from six pots were collected shortly after sampling. Soil samples were dried at 105℃ for 48 h in oven to determine the DW after determination of the FW. Soil water content (SWC) was calculated as follows: SWC (%) = [(FW − DW)/FW] × 100. Data are shown in Fig S1.

Determination of MDA and Proline
The content of malondialdehyde (MDA), the final product of lipid peroxidation, was measured as described by Du and Bramlage [29]. Proline content was determined according to the ninhydrin coloring method [30].

RNA Extraction and Real-Time PCR
The extraction of total RNA and the real-time polymerase chain reaction were performed based on the reference [31,32]. We designed real-time PCR primers using the Primer Express 3.0.1 (Table S1). The reaction consists of three biological replicates and three technical replicates. The 2 −ΔΔ Ct method was used for analyzing the fold change in gene expression levels, using Am18S as the reference.

Isoflavone Extraction and UHPLC-MRM-MS/MS Analysis
This part of the experiment was commissioned by Shanghai BioTree Biotech Co., Ltd (contract number: BQ-CYH20180626). The extraction of related compounds follows previous studies [33]. The instrument model is Agilent 1290 Infinity II series (Agilent Technologies), and the target compound was chromatographed using a Waters ACQUITY UPLC HSS T3 (100 × 2.1 mm, 1.8 μm, Waters) liquid chromatography column. The liquid phase A phase is 0.1% aqueous acetic acid, and B phase is methanol. The oven temperature was 40 °C, the sample tray was set to 4 °C, and the injection volume was 3 μL.
Mass spectrometry was conducted in a multiple reaction monitoring (MRM) mode using an Agilent 6460 triple quadrupole mass spectrometer equipped with an AJS-ESI ion source. The ion source parameters are as follows: capillary voltage = + 4000/ − 3500 V, nozzle voltage = + 500/ − 500 V, gas (N 2 ) temperature = 300 °C, gas (N 2 ) flow = 5 L/min, sheath gas (N 2 ) temperature = 250 °C, sheath gas flow = 11 L/min, and nebulizer = 45 psi. Several most sensitive transitions were used in the MRM scan mode to optimize the collision energy for each Q1/Q3 pair (Table S2).

Statistical Analysis
The data were analyzed using SPSS 19.0. All data are given as the mean and standard deviation of triplicate experiments. Treatment means were compared using Duncan's multiple range test. Pearson's correlations were calculated to measure the relationship among the variables.

Effects of Stress Treatments on Plant Growth
The seedlings of A. mongholicus were treated with drought for 15 days. As a result, about half of the drought-stressed plants exhibited leaf yellowing and curling. Shoot dry weight decreased to 0.20 g/plant on the 15th day, which were 66.4% of the control (Fig. 2a). Root dry weight decreased significantly to 0.09 g/plant on the 15th day, which were 83.6% of the control (Fig. 2b). After rewatering, shoot dry weight was only 51.7% of the control (Fig. 2a). Root dry weight was 71.0% of the control (Fig. 2b).

Accumulation of Metabolites Related to CG Synthesis in Different Organs Under Drought Stress
The content of compounds on the CG synthesis pathway was determined in different organs of A. mongholicus by UHPLC-MS/MS under drought stress, as shown in Fig. 3. Drought promotes the accumulation of all target compounds in the roots, except calycosin. The content of cinnamic acid, liquiritigenin, daidzein, and CG in roots were 0.1389 ± 0.0079 μg/g DW (dry weight), 0.2198 ± 0.0157 μg/g DW, 0.0643 ± 0.0050 μg/g DW, and 15.8522 ± 0.9069 μg/g DW, respectively, which were higher than other organs. L-phenylalanine and 4-coumaric acid were accumulated significantly in leaves after treatment. The contents of L-phenylalanine and 4-coumaric acid were 5.90 and 1.99 times higher than the control. Drought-induced accumulations of formononetin and calycosin in stems were 0.2632 ± 0.0154 and 0.6039 ± 0.0702 μg/g DW, respectively, which was 6.79 and 7.19 times over the levels in control plants.

Accumulation of Metabolites Related to CG Synthesis in Different Organs Under Rewatering
The contents of related compounds in different organs were detected under rewatering. As shown in Fig. 4, liquiritigenin, formononetin, and calycosin exhibited increased accumulation in response to rewater elicitation in roots, stems, and leaves of A. mongholicus. In Fig. 2 Effects of progressive drought stress and rewater on (a) shoot dry weight and (b) root dry weight of A. mongholicus. Red arrows indicate to start rewatering at the date. The data represent the mean values ± SD. The asterisk represents a significant difference (p < 0.05) contrast, the accumulation of cinnamic acid in stems and roots was decreased. Rewatering induced accumulation of daidzein in stems and the opposite trend appeared in leaves. As shown in Fig. 4d and Fig. 4e, the contents of liquiritigenin and daidzein in stems were 0.0748 ± 0.0028 μg/g DW and 0.0034 ± 0.0009 μg/g DW, which were 4.38 and 1.78 times higher than the control. More cinnamic acid, formononetin, calycosin, and CG were accumulated in roots than stems and leaves after rewatering. The contents of formononetin and calycosin in roots were significantly increased by 0.81 and 0.78 times compared with the control. In contrast, the content of cinnamic acid in roots was significantly decreased by 1.41 times compared with the control. However, no change in content of CG in roots is achieved compared with the control.

The Relative Expression Levels of Related Genes in Different Organs of A. mongholicus in Treatment Group
To investigate the expression levels of synthetic genes associated with target compound, we collected samples of roots, stems, and leaves. Quantitative real-time PCR was used to detect the expression of genes, and the enzymes encoded by these genes are involved in the synthesis of CG (Fig. 5). Compared to control, the expression of AmPAL, AmCHR, AmIFS, AmI3'H, and Am4CL of A. mongholicus was effectively enhanced in the roots, on the 15th day of drought. The expression levels of AmPAL, AmC4H, and Am4CL showed similar expression patterns under drought stress, wherein the expression level of AmPAL was increased by about threefold in the root. PAL, CHI, and IFS; catalytic synthesis of respective cinnamic acid; and liquiritigenin and daidzein in the CG pathway were upward expression levels in roots of A. mongholicus, just as the trends of these three compounds. After drought treatment, the expression of AmIOMT in A. mongholicus leaves significantly increased, and AmPAL, Am4CL, AmCHS, AmCHR, AmCHI, AmIFS, and AmI3'H decreased relative to the control. The expression of AmI3'H in the leaves decreased by 5 times.
After rewatering, that is the 21st day, all genes showed an increased trend in leaves, except AmCHR, AmCHI, and AmI3'H. PAL and C4H, catalytic synthesis of cinnamic acid, and 4-coumaric acid, respectively, were downward expression levels in stems of A. mongholicus with rewater treatment, just as the trends of cinnamic acid and 4-coumaric acid. Compared to the roots and stems, AmPAL, AmC4H, Am4CL, AmCHS, AmIFS, and AmIOMT all exhibited the highest expression levels in leaves, increasing by 3.17, 1.34, 4.98, 6.31, 3.57, and 2.42 times, respectively. The expression of AmPAL, AmC4H, AmIFS, and AmIOMT increased 1.24, 1.05, 1.02, and 1.14 times, respectively, in the roots, but the expression levels of these four genes in the stems decreased by 1.42, 1.82, 1.18, and 1.35 times, respectively. The expression levels of AmCHR, AmCHI, and AmI3'H were the highest in the stems compared with the other organs, increased by about 4.67, 4.36, and 3.11 times, respectively. The expression levels of these three genes in the leaves increased by 3.04, 1.29, and 1.33 times, respectively, while the expression levels of these genes in the roots decreased by 4.95, 1.03, and 1.33 fold, respectively.

Correlation Analysis of Target Metabolites and Related Enzyme Genes in CG Pathway
During this process, most of the metabolites and key enzyme genes related to the CG pathway changed. In order to clarify whether there was a correlation between these variables, correlation analysis of targeted metabolites and related genes in the leaves, stems, and roots were demonstrated. As shown in Fig. 6a, AmPAL and AmCHS have significant negative correlation with L-phenylalanine (p < 0.05). AmCHS also has a significant negative correlation with 4-coumaric acid and daidzein, in leaves (p < 0.05). AmI3'H in leaves showed significant positive correlation with calycosin (p < 0.05) but have Interestingly, AmPAL, AmCHI, AmIFS, and AmI3'H significantly affects the content of 4-coumaric acid (p < 0.01). AmCHI and AmI3'H have very significant negative correlation with daidzein (p < 0.01). AmIFS also exhibited a significant negative correlation with the accumulation of daidzein (p < 0.05) (Fig. 6a). In roots, AmIFS has significant positive correlation with daidzein (p < 0.05) but a significant negative correlation with formononetin (p < 0.05). Both AmPAL and AmCHI were significantly correlated with calycosin (p < 0.05). AmIFS has very significant negative correlation with calycosin (p < 0.01) (Fig. 6c).

Physiological Changes During Drought Acclimation
MDA is one of the lipid peroxidation products of plant cell membranes. The MDA contents in roots, stems, and leaves are increased drought stress. The MDA content in roots increased significantly from the 9th day, and it reached 3.54 times of the control by the 12th day. MDA levels in stems and leaves increased by 52% and 66%, respectively (Fig. 7a) on the 12th day, indicating more severe oxidative damage in the leaves. The content of proline in roots, stems, and leaves increased with the prolonged drought. The proline content increased slowly at the beginning of the drought and increased remarkably on the 12th day. On the 15th day, the proline contents in leaves, stems, and roots were 34.77, 54.27, and 36.29 times of that on the 0th day. On the 21th day, compared with the 15th day, the Fig. 6 Correlation map of target metabolites and relative genes in leaves (a), stems (b), and roots (c) of A. mongholicus in treatment group Each square indicates r (Pearson's correlation coefficient values for pairs of isoflavones or relative genes values). The red color represents a positive (0 < r < 1) correlation, and the blue color represents a negative (− 1 < r < 0) correlation. Single asterisk indicates that correlation is significant at the 0.05 level. Double asterisks indicate that correlation is significant at the 0.01 level content of proline in leaves, stems, and roots decreased by 72%, 81%, and 86%, respectively (Fig. 7b).

Discussion
In addition to causing material transfer between the shoot and root, the drought also increased the root-shoot ratio. Proline is the main organic osmotic agent, which plays an important role in the response of plants to abiotic stress, such as reducing the osmotic potential of cells to promote plant retention or absorption of water [34]. Previous studies have shown that Astragalus can increase proline levels under water-deficit stress [35]. In this study, the content of proline increased significantly with time under drought-stress treatment and reached the peak on the 15th day, and it was particularly higher in stems than in leaves. Drought stress led to lipid peroxidation, the production of MDA, and ultimately generated cell damage and plant death [36]. We observed that drought stress significantly increased the MDA content, reaching a peak on the 12th day, and the MDA content in roots was higher than that in leaves and stems (Fig. 7), indicating that the roots suffered more severe lipid peroxidation.
The biosynthetic pathway of flavonoids is one of the crucial secondary metabolic pathways in plants, and many key genes responding to environmental stress have been studied [37]. Under drought stress, AmPAL, AmC4H, and Am4CL showed similar expression patterns, elevated transcription levels in roots indicated that phenols in the roots were stimulated in response to drought, and these genes showed similar expression trends in most cases. Previous reports also highlighted the co-expression of these genes under drought conditions [37]. CHS is thought to be the entry point of the flavonoid pathway, where CHI converts chalcone to flavanone compounds, a prerequisite for many other flavonoid compounds [37]. CHS gene expression was the highest in the period of strong drought stress [38]. Previous studies found that water deficit can contribute to the expression of CHS in leaves and roots of Scutellaria baicalensis [39]. The expression of some gene overlaps under drought, salt stress, and low temperature stress [40]. Margarita et al. found that the expression of AmIFS in the leaves of Lotus japonicas was increased under drought treatment [41], while our study showed that the expression level of AmIFS in the leaves of A. mongholicus was decreased under drought treatment but increased significantly in the roots (Fig. 5). This may be due to different storage Fig. 7 Effects of progressive drought stress and hydration on leaves, stems, and roots. MDA content (a) and proline content (b) of A. mongholicus. Red arrows indicate to start rehydration at the date. Data represent the mean values ± SD. The asterisk represents significant difference (p < 0.05) organs of isoflavones, leading to various expression levels of AmIFS. The transcription levels of AmPAL, AmC4H, AmCHI, and AmIFS were upregulated in roots under drought induction and downregulated in leaves and stems. This change is similar to the accumulation pattern of the corresponding compounds of these enzymes. For example, the isoflavones of Lotus japonicus are mainly stored in the leaves [42], while the isoflavones of A. mongholicus are mainly stored in the roots. AmIOMT is highly expressed in the context of water deficiency, which has similar results in chickpeas (Cicer arietinum L.) [43]. It has been reported that the expression level of I3'H was increased significantly in response to drought, low temperature, and salt stress [44]. I3'H in roots of A. mongholicus was highly expressed in response to drought stress (Fig. 5). These results show that high expression of IFS and I3'H contributes to the accumulation of CG in roots after drought stress.
In this study, drought stress exhibited an obvious induction effect on the accumulation of isoflavones in A. mongholicus (Fig. 3). There was a significant negative correlation between AmIFS and the accumulation of daidzein in leaves, but it has a significant positive correlation with daidzein in roots (Fig. 6). This may indicate that daidzein is synthesized in the leaves and transported to the roots. The expression level of AmIOMT in the leaves was the highest under drought stress, while the product of IOMT catalyzed synthesis, formononetin, was accumulated mostly in the stem (Fig. 3,  Fig. 5). One possible origin is that significant increased AmIOMT expression level in leaves, leading to a large amount of formononetin synthesis in leaves, which is then transported to the stem through the petiole. This is consistent with previous reports. Isoflavones were found in the secretion of phloem of Astragalus membranaceus petiole [45]. Another reason is probably that AmIOMT interferes or even blocks the process of regulating and synthesizing formononetin during water deficiency. The accumulation of calycosin and CG in leaves was significantly reduced drought stress compared with the control one, indicating that formononetin might be transported to other organs instead of being transformed into downstream metabolites in leaves [46]. Previous evidence has demonstrated that under drought stress, the accumulation of CG, an active ingredient with medicinal value, in Astragalus membranaceus root increased drought stress [28,35], which probably results from calycosin, a precursor compound synthesized from CG in stem [31,45].

Conclusions
Previous studies mainly focused on the influences of abiotic stress on CG content, which is the final metabolite content in the calycosin-7-O-β-D-glycoside (CG) synthesis pathway, while the content of all intermediate metabolites in this pathway was measured in this research. Moreover, the regulation mechanism of CG accumulation in different organs under continuous drought conditions was studied. The accumulation of calycosin and CG in roots under drought stress may be derived from formononetin synthesized in leaves. This study explored the flavonoid synthesis of plants undergoing abiotic stress on the level of metabolites and key enzyme gene expression, which is of great significance for obtaining high-quality Astragalus membranaceus in arid or semi-arid area. In traditional Chinese medicine, the root of Astragalus membranaceus is used, and most of its stems and leaves are discarded. Therefore, the future research will focus on the comprehensive utilization of the non-medicinal parts of Astragalus species.