Special Genotypes of ABA Key Functional Genes NCEDs Members in the Glycyrrhizic Acid Synthesis Regulatory Network Promote Glycyrrhizic Acid Synthesis in Licorice


 BackgroundThe glycyrrhizic acid biosynthesis pathway does not exist in isolation, but is connected with the biosynthesis pathways of other secondary metabolites in licorice and finally forms a network. Our previous study found that exogenous spraying of appropriate concentration of abscisic acid (ABA) could increase the content of glycyrrhizic acid (GA) in licorice. However, the mechanism of action remains unknown. We aim to understand the molecular mechanism of ABA promoting the synthesis of GA in licorice and find the molecular marker for the high content of GA germplasm material.MethodsWe analyzed the expression of the key gene of β-AS for GA synthesis after applying ABA, the key functional genes NCED1, NCED3 and NCED4 in the process of ABA synthesis were overexpression, and analyzed the relationship between the SNP polymorphism of the NCED1, NCED3, NCED4 and the content of the GA and ABA in 13 different provenances of licorice with the grey correlation analysis. ResultsThe appropriate concentration of ABA treatment could increase the content of the GA through improving the expression of β-AS. There were significant differences in the content of ABA and GA among the 13 provenances, and the 3 members of the NCEDs family of different provenances had abundant SNP variation sites. Grey correlation and overexpression of NCEDs function both showed that the effect of promoting the synthesis of ABA and GA: NCED1 437 bp G type > NCED3 966 bp G type > NCED4 845 bp A type. All of the above indicated that NCEDs gene variation was the reason for the diversity of GA and ABA content. When selecting high GA germplasm, more priority should be given to NCED1 gene 437 bp G type, NCED3 gene 966 bp G type and NCED4 gene 845 bp A type. ConclusionThis study provides a basis for the selection of excellent GA content germplasm of licorice, and provide some reference for producing a high quality cultivated licorice.


Introduction
Licorice is the most important bulk medicinal material, and it is also an important additive in cosmetics, health care products, tobacco and other industries. The annual demand is huge [1]. Modern pharmacology has proved that the licorice and its extract has anti-in ammatory and antiviral antiulcer, antiin ammation, spasmolysis, antioxidative, contravariance, antiviral, anticancer activities, hepatoprotective, eliminating phlegm and reinforcing remembrance effects [2]. Triterpene saponins are the main chemical components of licorice [3] , which mainly include glycyrrhizic acid (GA) and glycyrrhetinic acid. GA is more popular than glycyrrhetinic acid, which can exhibit potent biological NCEDs members among different germplasms of licorice? Can the polymorphism of NCEDs members promote the accumulation of ABA and GA?
Based on this, the main objectives of this study are as follows: 1) The expression of functional genes for GA synthesis after ABA treatment. 2) Starting from the substitution network that promotes the synthesis of GA, the key genes of ABA endogenous regulatory substances NCEDs were cloned, analyzed, veri ed and treated with inducing factors, and the mechanism of promoting GA synthesis was analyzed. 3) Through the analysis of the variation of ABA content in different germplasms of licorice, the status of ABA in different germplasms under natural state could be understand. Through decomposing the correlation between the polymorphism of the key functional genes of ABA synthesis and the content of ABA and GA, analyze the molecular mechanism of the variation of ABA key functions affecting the differential formation of GA could be analyzied. This study provided the basis for explaining the difference of GA synthesis e ciency of different provenances, and supported for the selection of licorice germplasm with high GA content.

Materials
The seeds of different provenances was collected from 13 main producing areas of licorice in China (marked as YX,HT,AH,CF,JQ,HJ1,HJ2,ZZ,ETK,GD,YC,MQ,KEL). The information was shown in Table 1. They were sown in the Medicinal Botanical Garden of Beijing University of Chinese Medicine and harvested one and a half years later. The licorice used to extract RNA and DNA were sown in 15cm × 12cm owerpots (Sand: Vermiculite = 1:1) and cultivated in an arti cial climate incubator with a temperature of 25 ± 2 ℃ and a light time of 14 h/d.

The effect of ABA treatment on the expression of key functional gene β-AS in licorice
The licorice was treated with different concentrations of ABA after 60 days of growth from seeds, the concentrations were 25, 50, 100 and 200 mg/L respectively, each treatment was repeated three times, and pure water was used as the control group. Samples were taken at 3, 6, 12 and 24 hours of treatment, 90 mg root samples were frozen in liquid nitrogen and stored at -80 ℃. Then RNA was extracted according to RNA Extraction Kit (Biomed, Beijing). After extraction, reverse transcription was performed. Reverse transcription reaction system: 10 μL of the last reaction solution, 1 μL of primer script RT enzyme mix, 1 μL of RT primer mix, 4 μL of 5 × primer script buffer, 4 μL of RNase free water, the total system was 10 μL (The primer sequences were in Supplementary Materials Table 2 The licorice that grows for one and a half years was collected. The determination of ABA on the root tip of licorice was measured with ELISA kit, referring to the instruction manual for speci c operation. The method for determining the effective components of licorice was as follows: 0.15 g of constant weight licorice powder was weighed, then it was putted into a 100 mL conical ask with stopper, 40 mL 70% ethanol was added and weighed it tightly. It was treated with ultrasound for 30 min, maked up for the weight loss with 70% ethanol, shaked it well and passed through 0.45 μm lter membrane for standby. An appropriate amount of GA reference substances were accurately weighed, and added with methanol to prepare a mixed reference solution with GA 110.80 μg/mL. The chromatographic conditions were as follows: dual wavelength HPLC detection. Mobile phase: acetonitrile (A) -0.05% phosphoric acid water (B); gradient elution: 0 ~ 8 min, 20% A; 8 ~ 30 min, 20% ~ 35% A; 30 ~ 42 min, 35% ~ 45% A. Detection wavelength: 237nm for GA. The ow rate was 1.0 mL/min, the column temperature was 30 ℃, and the injection volume was 10 μL. Each sample was repeated three times.

Cloning of NCEDs from licorice
The whole plant of licorice growing for 30 days was used to extract the total RNA with the plant total RNA Extraction Kit (Biomed, Beijing), and then reverse transcription was performed to synthesize cDNA. Primers were designed according to the homologous regions of the reported NCED genes (NCED1, NCED3, NCED4), and 3 'and 5' RACE. The full-length primer sequences of the genes were shown in Supplementary Materials Table 3, Table 4, Table 5. The open reading frame of NCEDs was ampli ed.
The primers were connected to pMD19T (pMD-NCED1, pMD-NCED3, pMD-NCED4) vector and transformed into E. coli. The positive single colony was selected and sequenced.

Analysis of the speci c expression of NCEDs members in licorice
The root treated with PEG at concentrations of 0%, 5%, 10% and 15% were used for RNA extraction. After reverse transcription was completed, real-time PCR was performed. The Real Time PCR conditions were as follows: pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 10 s, annealing at 57 ℃ for 30 s, extension at 72 ℃ for 30 s, 40 cycles. The primer sequence was shown in Supplementary Materials Table 6. Each experiment was repeated three times.

SNPs analysis of NCEDs genes of licorice from different provenances
The DNA of licorice was extracted from leaves of licorice. According to the full-length sequence of NCED1, NCED3 and NCED4, primers were designed to amplify the three genes in licorice. The PCR reaction system was 30 μL. the reaction conditions were: 94℃ pre denaturation for 5 min, 94℃ denaturation for 30 s, annealing at 55℃ for 1 min, 72℃ extension for 2 min, 40 cycles, 72℃ extension for 10 min.

Overexpression of NCEDs gene
Tobacco TobRB7 promoter was designed and synthesized according to NCBI sequence. Two restriction sites of Sal I and Bgl II were selected to linearize the vector pCAMBIA1305.1, Sal I and Bgl II double-digested TobRB7, and the T4 ligase method was used to ligate it into the vector to generate pCAMBIA1305.1-TobRB7. The pCAMBIA1305.1-TobRB7 was linearized by Bgl II and Spe I restriction enzymes. The NCED1, NCED3, and NCED4 were cloned by Bgl II and Spe I restriction enzymes and the recombinant plasmid was obtained. The recombinant plasmid was transformed into E. coli. The cloned recombinant plasmid was extracted with high-purity plasmid small-scale rapid extraction kit (Biomed, Beijing), and transformed into Agrobacterium tumefaciens EHA105 by electric shock transformation. Synthesize EHA-TobRB7-NCED1, EHA-TobRB7-NCED3, EHA-TobRB7-NCED4 engineering bacteria were synthesized. A single colony of engineered bacteria was inoculated into YEB liquid medium and cultured at 28°C and 280 rpm. The seeds of licorice were soaked in the bacterial solution, cultured for 24 hours and then changed to sterile water for culture and stained with GUS (GUS staining blue proved that the engineered bacteria successfully infects licorice, and the target gene was expressed transiently). The seedlings were infected with EHA-TobRB7-NCED1, EHA-TobRB7-NCED3, EHA-TobRB7-NCED4. After the rst dection on GUS, the roots were removed to test the content of GA, ABA and β-AS content.

Data analysis
All experiments were repeated three times. GA content and ABA content were expressed as mean ± standard deviation, SPSS22.0 software was used for analysis and ANOVA was used. P < 0.05 was considered to have signi cant difference. P < 0.01 was considered to have extremly signi cant difference. MATLAB7.0 software was used to analyze the relationship between SNPs of NCEDs gene and GA content. The values of G, A, T and C were assigned to 1, 2, 3, and 4 respectively to ensure the elimination of dimensions and the accuracy of modeling. The content of GA was different among different provenances. YX has the highest GA content. There was no signi cant difference in GA content between AH and CF and YX. MQ has the lowest GA content ( Fig.  2A). There were also differences in ABA content among different provenances. The ABA content of HJ2 was the highest, which has no signi cant difference with GD. HT contained the lowest ABA (Fig. 2B). The differences in GA and ABA content within each provenance provide favorable materials for further study of the variation of GA and ABA content.

Cloning and functional veri cation of NCEDs
The full length of the open reading frames of NCED1, NCED3, and NCED4 were 1830 bp, 1830 bp and 1764 bp, respectively. All three genes had one exon and no intron (supplementary material). The three NCED genes were compared and found to have high similarities with the NCED gene sequences of various plants ( Figure 3A, 3B, 3C; supplementary materials). They can encode 609, 609 and 587 amino acids, respectively. The molecular weights of the hypothetical proteins were 67.56 kDa, 67.29 kDa and 63.98 kDa, respectively, and their isoelectric points were 6.64, 5.95, and 6.26, respectively. After treating licorice with different concentrations of PEG to simulate the effect of drought on the ABA content of licorice and the relative expression of NCEDs gene family members ( Figure 3D), the results showed that the ABA content increased with the increase of PEG concentration, 10% and 15% had signi cantly differences compared with the control group. That is, drought stress could promote the increase of ABA content of licorice. As the concentration of PEG increased, the relative expression levels of the three genes all showed an increasing trend ( Figure 3E). That is, drought stress can signi cantly increase the relative expression of NCEDs gene family members. At the same time, the expression of NCED3 gene was signi cantly higher than the other two genes when treated with high concentration, indicating that this gene has a stronger effect under severe drought conditions.

SNPs analysis of NCEDs genes of licorice from different provenances
Among the 13 provenance samples, the numbers of NCED1, NCED3, and NCED4 samples were successfully cloned and sequenced, respectively, 65, 67, and 36.  Table 9). Among them, the genotype with the largest number of samples is NCED-G1, with a total of 9 samples. There are more genotypes for 3 genes and 1 provenance. Since some provenances were distributed in different genotype lengths, it showed that there were rich variations within the provenances, while some genotypes contain samples from different provenances, which showed that there was consistency between different provenances.

Correlation analysis between SNPs of NCEDs and GA content
Among all SNPs of NCED1, NCED3 and NCED4, mutation sites with a correlation coe cient greater than 0.8 for GA content accounted for 78.57%, 76.92%, and 72.14% of the corresponding gene mutation sites (Supplementary Materials 13, 14, 15), indicating three The mutant gene of the patient has a strong promoting effect on the synthesis and accumulation of GA, and the mutation site of NCED1 gene has a better effect on the synthesis and accumulation of ABA during the sampling period than the other two genes. The highest correlation between NCED1 gene and GA content is the SNP site (G/A) at 437 bp with a correlation coe cient of 0.8779 (Supplementary Material Table 10). Its G type is more conducive to the synthesis and accumulation of GA. NCED3 gene and NCED1 have similar results (Supplementary  Materials Table 11). Its G type is more conducive to the synthesis and accumulation of GA. The highest correlation between NCED4 gene and GA content is the SNP locus at 845 bp (A/G) with a correlation coe cient of 0.8762 (Supplementary Table 12). Its type A is more conducive to the synthesis and accumulation of GA. These results indicate that the above three loci are the loci that have the greatest impact on GA synthesis and accumulation among the three genes.

Revalidation of NCEDs
After treatment with the engineered bacteria, the expression of β-AS gene showed a trend of increase ( Figure 4A). There were signi cant differences in the expression of β-AS gene between the licorice infected by different engineered bacteria and the licorice of the control group. The degree of improvement was EHA-TobRB7-NCED1> EHA-TobRB7-NCED3> EHA-TobRB7-NCED4. Therefore, the G-type at 437 bp of NCED1 gene sequence had a stronger effect on the increase of β-AS gene expression during this sampling period than NCED3 and NCED4, but these two also had a certain effect on the accumulation of β-AS gene expression. In addition, the results of the ABA and GA content of the seedlings treated with the engineered bacteria showed that NCED1 promoted the ABA and GA content better than the other two genes ( Figure 4B, 4C). In short, the G type at 437 bp of NCED1 gene sequence has a stronger in uence on the synthesis and accumulation of GA and ABA during the sampling period than NCED3 and NCED4, but the two also have a certain in uence on the synthesis and accumulation of GA and ABA [26].

Discussion
ABA is an important connection between the plant's response to adversity and secondary metabolites, and the secondary metabolites produced can protect plants from environmental stress, so it can promote the synthesis of certain secondary metabolites. When seedlings were treated with different concentrations of ABA, it was found that low concentrations (25-50 mg/L) of ABA could continuously promote the increase of the relative expression of β-AS during the synthesis of GA. Therefore, low concentration promoted the increase of GA content by promoting the expression of key genes in the process of GA synthesis.
After planting licorice from wild provenances in 13 major production areas for one and a half years, the GA content of licorice was measured. It was found that signi cant differences existed not only among the provenances, but also within the provenances. These were mainly determined by the the complex genetic background of licorice, which was consistent with previous studies [27]. Phytohormones play an extremely important role in the growth and development of plants, which are mainly manifested by signal transduction to enable plants to adapt to external stimuli and promote their own development [16] [28]. The main reason for the insigni cant differences in ABA between different provenances was the sampling period, but the larger differences within the same provenance were consistent with the predecessors, which also showed that the genetic background of licorice was more complicated.
NCEDs are one of the key functional genes in the process of ABA synthesis, which play a major role in the process of ABA synthesis. Three members of the NCEDs gene family were cloned from licorice by using a combination of homologous cloning and RACE methods. Bioinformatics analysis found that these three genes had a homology of more than 70% with the corresponding genes of other plants in the same family. After simulated drought stress, the relative expression of the three genes increased in synchrony with the increase in ABA and β-AS.
Among the 43 genotypes NCED1, NCED3, and NCED4, the highest correlation coe cients with GA were at 437 bp in NCED1 gene, 966 bp in NCED3 gene, 845 bp in NCED4 gene, and the mutation sites with highest correlation coe cient with ABA were 755 bp in NCED1 gene, 443 bp in NCED3 gene, 1645 bp in NCED4 gene, and the order of the correlation coe cient between GA and the 3 genes with different mutation sites was not consistent with the order of the correlation coe cient between ABA and the 3 genes with different mutation sites. The possible reason was that the appropriate concentration of ABA can promote the synthesis and accumulation of GA, while the high concentration of ABA inhibits the synthesis and accumulation of GA according to our results.
Due to the existence of degenerate codons, the mutation of functional gene SNP has two results: synonymous mutation and missense mutation. However, these two results play a certain role in promoting the synthesis and accumulation of GA and ABA in the association analysis. Missense mutations can affect the catalytic e ciency of proteins to a certain extent by changing the types of amino acids. For example, in NCED3, the correlation coe cient between 4 missense mutation sites and GA content in 6 missense mutation sites was greater than 0.8 and was located in the rst 4 positions of all mutation coe cients. It showed that the missense site was the reason for changing the catalytic e ciency of the enzyme. But there were also cases where missense mutations have less effect, while synonymous mutations had a greater effect. Synonymous mutations may change the helical structure of the DNA sequence through the difference in the charge number of different bases, thus promoting the synthesis and accumulation of certain substances [29].
The biosynthetic pathway of glycyrrhizic acid does not exist in isolation. It is connected with other secondary metabolites such as ABA, GA, glycyrrhizin, glycyrrhizin and other biosynthetic pathways to form a network. NCEDs genes are one of the rate-limiting enzymes in the ABA synthesis pathway, and its expression directly affects the synthesis of ABA and then the activity of glycyrrhizic acid biosynthesis enzymes, regulating the synthesis of GA.
This study showed that appropriate concentration of ABA treatment can increase the expression of key functional gene β-AS in the process of GA synthesis. The NCEDs family members NCED1, NCED3 and NCED4, the key functional genes of ABA, could promote the synthesis of ABA. We collected 115 samples from 13 wild provenances of licorice from different provenances, analyzed their GA and ABA content, and found that the GA and ABA content of different provenances and different individual plants had large variations. More importantly, after the grey correlation analysis between the SNPs of the three genes and the content of ABA and GA, the three NCED family genes were re-veri ed. The NCED1 gene variant sites were compared to the total synthesis and accumulation of ABA and GA during the sampling period. The role of NCED1 is better than that of NCED3 and NCED4, but the two genes of NCED3 and NCED4 also have a certain effect on the synthesis and accumulation of ABA and GA, that is, NCED1 gene 437 bp G type > NCED3 gene 966 bp G type > NCED4 gene 845 bp A type. Therefore, in the selection of high glycyrrhizic acid germplasm, priority should be given to the G type at 437 bp in NCED1 gene, G type at 966 bp in NCED3 gene and A type at 845 bp in NCED4 gene.
These studies have created a new idea: to explain the mechanism of the variation of the regulated end product from the variation of the key regulatory substances in the regulatory network to explain the reasons for the variation of the GA content of different genotypes, and to provide a more direct standard for the selection of high-quality licorice germplasm.

81773838) Availability of data and materials
The data used and/or investigated during the present study are available from the corresponding author upon reasonable request.

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Medicine. Figure 1 Effects of applying different concentrations of ABA on the relative expression of key functional gene β-AS