Effects of silicon application on leaf structure and physiological characteristics of Glycyrrhiza uralensis Fisch. and Glycyrrhiza inflata Bat. under salt treatment.

BACKGROUND: Soil salinization leads to a significant decline in crop yield and quality, including licorice, an important medicinal cash crop. Studies have proofed that the application of exogenous silicon can significantly improve the ability of licorice to resist salt stress, however, few studies concentrated on the effects of foliar silicon application on the morphology, physiological characteristics, and anatomical structure of licorice leaves under salt stress. In this study, the effects of Si (K2SiO3) on the structural and physiological characteristics of Glycyrrhiza uralensis Fisch. and G. inflata Bat. leaves under different salt concentrations (medium- and high-salt) were studied. RESULTS: Compared with the control (without salt), the plant height, total dry weight, leaf area, leaf number, relative water content, xylem area, phloem area, ratio of palisade to spongy tissue, gas exchange parameters, and photosynthetic pigment content of both licorice varieties were significantly reduced under high-salt (12S) conditions. However, the thickness of the leaf, palisade tissue, and spongy tissue increased significantly. Applying Si to the leaf surface increased the area of the vascular bundle, xylem, and parenchyma of the leaf's main vein, promoted water transportation, enhanced the relative leaf water content, and reduced the decomposition of photosynthetic pigments. These changes extended the area of photosynthesis and promoted the production and transportation of organic matter. G. uralensis had a better response to Si application than did G. inflata. CONCLUSIONS: In conclusion, foliar application of Si can improve water absorption, enhance photosynthesis, improve photosynthetic capacity and transpiration efficiency, promote growth and yield, and alleviate the adverse effects of salt stress on the leaf structure of the two kinds of licorice investigated.

Root-associated endophytic bacterial community composition and structure of three medicinal licorices and their changes with the growing year.

BACKGROUND: The dried roots and rhizomes of medicinal licorices are widely used worldwide as a traditional medicinal herb, which are mainly attributed to a variety of bioactive compounds that can be extracted from licorice root. Endophytes and plants form a symbiotic relationship, which is an important source of host secondary metabolites. RESULTS: In this study, we used high-throughput sequencing technology and high-performance liquid chromatography to explore the composition and structure of the endophytic bacterial community and the content of bioactive compounds (glycyrrhizic acid, liquiritin and total flavonoids) in different species of medicinal licorices (Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata) and in different planting years (1-3 years). Our results showed that the contents of the bioactive compounds in the roots of medicinal licorices were not affected by the species, but were significantly affected by the main effect growing year (1-3) (P < 0.05), and with a trend of stable increase in the contents observed with each growing year. In 27 samples, a total of 1,979,531 effective sequences were obtained after quality control, and 2432 effective operational taxonomic units (OTUs) were obtained at 97% identity. The phylum Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, and the genera unified-Rhizobiaceae, Pseudomonas, Novosphingobium, and Pantoea were significantly dominant in the 27 samples. Distance-based redundancy analysis (db-RDA) showed that the content of total flavonoids explained the differences in composition and distribution of endophytic bacterial communities in roots of cultivated medicinal liquorices to the greatest extent. Total soil salt was the most important factor that significantly affected the endophytic bacterial community in soil factors, followed by ammonium nitrogen and nitrate nitrogen. Among the leaf nutrition factors, leaf water content had the most significant effect on the endophytic bacterial community, followed by total phosphorus and total potassium. CONCLUSIONS: This study not only provides information on the composition and distribution of endophytic bacteria in the roots of medicinal licorices, but also reveals the influence of abiotic factors on the community of endophytic bacteria and bioactive compounds, which provides a reference for improving the quality of licorice.

Calcium-Dependent Protein Kinase Genes in Glycyrrhiza Uralensis Appear to be Involved in Promoting the Biosynthesis of Glycyrrhizic Acid and Flavonoids under Salt Stress.

As calcium signal sensors, calcium-dependent protein kinases (CPKs) play vital roles in stimulating the production of secondary metabolites to participate in plant development and response to environmental stress. However, investigations of the Glycyrrhiza uralensis CPK family genes and their multiple functions are rarely reported. In this study, a total of 23 GuCPK genes in G. uralensis were identified, and their phylogenetic relationships, evolutionary characteristics, gene structure, motif distribution, and promoter cis-acting elements were analyzed. Ten GuCPKs showed root-specific preferential expressions, and GuCPKs indicated different expression patterns under treatments of CaCl2 and NaCl. In addition, under 2.5 mM of CaCl2 and 30 mM of NaCl treatments, the diverse, induced expression of GuCPKs and significant accumulations of glycyrrhizic acid and flavonoids suggested the possible important function of GuCPKs in regulating the production of glycyrrhizic acid and flavonoids. Our results provide a genome-wide characterization of CPK family genes in G. uralensis, and serve as a foundation for understanding the potential function and regulatory mechanism of GuCPKs in promoting the biosynthesis of glycyrrhizic acid and flavonoids under salt stress.

Silicon improves salt tolerance of Glycyrrhiza uralensis Fisch. by ameliorating osmotic and oxidative stresses and improving phytohormonal balance.

Si has a beneficial effect on improving plant tolerance to salt stress. Nevertheless, the mechanisms of Si in mediating the stress responses are still poorly understood. Glycyrrhiza uralensis Fisch. (G. uralensis), a well-known medicinal plant, possesses vast therapeutic potentials. In the present study, a pot experiment was conducted to investigate the long-term effects of Si on growth and physiobiochemical characteristics in 2-year-old G. uralensis subjected to different levels of salinity. Si markedly affected G. uralensis growth in a salt concentration-dependent manner and had no effect on G. uralensis growth under 6 g/kg NaCl. However, it partly reversed the reduction effect induced by 9 g/kg NaCl. In addition, Si significantly increased the contents of soluble sugar and protein but deceased proline content and thus increased water relations; Si markedly increased the activities of SOD, peroxidase, and CAT and further resulted in decreased MDA content and membrane permeability. Moreover, Si altered the levels of phytohormones and their balances. With correlation analysis and principal component analysis (PCA), root biomass had a significant negative correlation with MDA and membrane permeability while a positive correlation with indole-3-acetic acid and GA3. The PCA partitioned the total variance into three PCs contributing maximum (88.234%) to the total diversity among the salt stress with or without Si due to the study of various traits. In conclusion, Si exerts a beneficial property on salt-induced harmful effects in G. uralensis by relieving osmotic stress, improving water relations, and alleviating oxidative stress; thus, altering the levels and balance of phytohormones results in improved growth of salt-stressed G. uralensis.

The Basic Helix-Loop-Helix Transcription Factor GubHLH3 Positively Regulates Soyasaponin Biosynthetic Genes in Glycyrrhiza uralensis.

Glycyrrhiza uralensis (licorice) is a widely used medicinal plant belonging to the Fabaceae. Its main active component, glycyrrhizin, is an oleanane-type triterpenoid saponin widely used as a medicine and as a natural sweetener. Licorice also produces other triterpenoids, including soyasaponins. Recent studies have revealed various oxidosqualene cyclases and cytochrome P450 monooxygenases (P450s) required for the biosynthesis of triterpenoids in licorice. Of these enzymes, ß-amyrin synthase (bAS) and ß-amyrin C-24 hydroxylase (CYP93E3) are involved in the biosynthesis of soyasapogenol B (an aglycone of soyasaponins) from 2,3-oxidosqualene. Although these biosynthetic enzyme genes are known to be temporally and spatially expressed in licorice, the regulatory mechanisms underlying their expression remain unknown. Here, we identified a basic helix-loop-helix (bHLH) transcription factor, GubHLH3, that positively regulates the expression of soyasaponin biosynthetic genes. GubHLH3 preferentially activates transcription from promoters of CYP93E3 and CYP72A566, the second P450 gene newly identified and shown to be responsible for C-22ß hydroxylation in soyasapogenol B biosynthesis, in transient co-transfection assays of promoter-reporter constructs and transcription factors. Overexpression of GubHLH3 in transgenic hairy roots of G. uralensis enhanced the expression levels of bAS, CYP93E3 and CYP72A566. Moreover, soyasapogenol B and sophoradiol (22ß-hydroxy-ß-amyrin), an intermediate between ß-amyrin and soyasapogenol B, were increased in transgenic hairy root lines overexpressing GubHLH3. We found that soyasaponin biosynthetic genes and GubHLH3 were co-ordinately up-regulated by methyl jasmonate (MeJA). These results suggest that GubHLH3 regulates MeJA-responsive expression of soyasaponin biosynthetic genes in G. uralensis. The regulatory mechanisms of triterpenoid biosynthesis in legumes are compared and discussed.

Effect of Abscisic Acid on Accumulation of Five Active Components in Root of Glycyrrhiza uralensis.

Licorice is one of the most generally used herbal medicines in the world; however, wild licorice resources have decreased drastically. Cultivated Glycyrrhiza uralensis Fischer are the main source of licorice at present, but the content of main active components in cultivated G. uralensis are lower than in wild G. uralensis. Therefore, the production of high-quality cultivated G. uralensis is an urgent issue for the research and production fields. In this study, the content of five active components and seven endogenous phytohormones in cultivated G. uralensis (two-year-old) were determined by high-performance liquid chromatography (HPLC) and enzyme-linked immunosorbent assay (ELISA), respectively. Furthermore, different concentrations (25-200 mg/L) of exogenous abscisic acid (ABA) were sprayed on the leaves of G. uralensis in the fast growing period. Results showed that ABA, zeatin riboside (ZR), and dihydrozeatin riboside (DHZR) had strong correlation with active components. In addition, the content of five active components increased remarkably after ABA treatment. Our results indicate that ABA is significantly related to the accumulation of active components in G. uralensis, and the application of exogenous ABA at the proper concentration is able to promote the accumulation of main components in G. uralensis.

Silicon alleviates salt and drought stress of Glycyrrhiza uralensis seedling by altering antioxidant metabolism and osmotic adjustment.

This study was conducted to determine effect and mechanism of exogenous silicon (Si) on salt and drought tolerance of Glycyrrhiza uralensis seedling by focusing on the pathways of antioxidant defense and osmotic adjustment. Seedling growth, lipid peroxidation, antioxidant metabolism, osmolytes concentration and Si content of G. uralensis seedlings were analyzed under control, salt and drought stress [100 mM NaCl with 0, 10 and 20% of PEG-6000 (Polyethylene glycol-6000)] with or without 1 mM Si. Si addition markedly affected the G. uralensis growth in a combined dose of NaCl and PEG dependent manner. In brief, Si addition improved germination rate, germination index, seedling vitality index and biomass under control and NaCl; Si also increased radicle length under control, NaCl and NaCl-10% PEG, decreased radicle length, seedling vitality index and germination parameters under NaCl-20% PEG. The salt and drought stress-induced-oxidative stress was modulated by Si application. Generally, Si application increased catalase (CAT) activity under control and NaCl-10% PEG, ascorbate peroxidase (APX) activity under all treatments and glutathione (GSH) content under salt combined drought stress as compared with non-Si treatments, which resisted to the increase of superoxide radicals and hydrogen peroxide caused by salt and drought stress and further decreased membrane permeability and malondialdehyde (MDA) concentration. Si application also increased proline concentration under NaCl and NaCl-20% PEG, but decreased it under NaCl-10% PEG, indicating proline play an important role in G. uralensis seedling response to osmotic stress. In conclusion, Si could ameliorate adverse effects of salt and drought stress on G. uralensis likely by reducing oxidative stress and osmotic stress, and the oxidative stress was regulated through enhancing of antioxidants (mainly CAT, APX and GSH) and osmotic stress was regulated by proline.

[Effect of exogenous brassinolide on morphological characters and contents of seven chemical constituents of Glycyrrhiza uralensis].

The transplants of the two-year-old Glycyrrhiza uralensis were subjected to four concentration of brassinolide (BR 0.1, 0.4, 0.7, 1.0 mg•L⁻¹) in July. The morphological characters ( plant height, stem diameter, nodes number, internode length and root length , root thick, root fresh weight and root dry weight ) were measured and seven kinds of chemical constituents (glycyrrhizic acid, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, liquiritin apioside, isoliquiritin apioside) were determined by HPLC with the aim of increasing sinter output and improving quality of G. uralensis. Then the long-term dynamic changes of these morphological characters and chemical compositions' content were analyzed. The results showed that morphological characters of plant height, stem diameter, root length , root thick, root fresh weight and root dry weight increased remarkably with the 0.7 mg•L⁻¹ BR stimulating 2 months later,the increase rates were： 15.09%,6.15%,16.52%,8.46%,21.90%,29.41%, respectively. The content of glycyrrhizic acid, liquiritin, isoliquiritin, liquiritigenin, liquiritin apioside, isoliquiritin apioside were increased 20.16%,45.31%,53.56%,27.66%,23.54%,8.46% with the 0.7 mg•L⁻¹ BR stimulating 2 months later. The best effects were achieved in 2 months after brassinolide stimulating. The conclusions prove that morphological characters and the main chemical constituents accumulation of G. uralensis could be effected by exogenous BR stimulation in certain case.

[Effects of abscisic acid on chemical components content and color of Glycyrrhiza uralensis].

An experiment was conducted using cultivated Glycyrrhiza uralensis in age of one year to study the effects of abscisic acid (ABA) on chemical components content and color of G. uralensis. By using different concentrations of ABA spraying on leaves, the change of the chemical component content was analyzed within 45 d after ABA stimulation, and the effects on quality were studied combined with colorimetric analysis data. It turned out that in some sense the content of glycyrrhizic acid and liquiritin had increased within 45 d, especially for liquiritin. After high concentrations of ABA (3.96 mg · L(-1)) stimulating, the content of glycyrrhizic acid rose 52% while liquiritin up 392% within 30 d. Then they both showed a decline in the content of glycyrrhizic acid and liquiritin on 45 d. Color index values of a* and b* were all significantly higher than that of the control group within 45 d, which meant the color of powders turned toward red and yellow. The conclusion was that ABA (3.96 mg · L(-1)) stimulating could not only improve the quality in the traditional sense through the color of G. uralensis, but also in the modern sense by improving the content of glycyrrhizic acid and liquiritin.

[Interaction relationship between secondary metabolites in Glycyrrhiza uralensis at condition of short-term exogenous glycyrrhizic acid simulation].

In order to study the interaction relationship between secondary metabolites in Glycyrrhiza uralensis, and find out which secondary metabolite is significantly related to the content of glycyrrhizic acid, artificial applying ammonium glycyrrhetate solution was used to establish a high glycyrrhizic acid environment. The change of the 4 secondary metabolites was analyzed within 72 h after glycyrrhizic acid stimulation, while correlation statistical soft was applied to analyze the correlation of glycyrrhizic acid and other compositions. It turned out that it is feasible to establish high glycyrrhizic acid environment by glycyrrhizic acid root soaking in the concentration of 1.0 mmol x L(-1). There was significant positive correlation between glycyrrhizic acid and liquorice glycosides in short-term glycyrrhizic acid stimulation environment. It is concluded that glycyrrhizic acid accumulation internal of G. uralensis could be effected by artificial exogenous glycyrrhizic acid stimulation in certain case, and its accumulation was significantly related to the content of liquorice glycosides.

[Effect of nitrogen supply on biomass accumulating and root respiration dynamic changing of Glycyrrhiza uralensis].

This paper aimed to study the effect nitrogen supplying on biomass accumulation and root respiration dynamic change of Glycyrrhiza uralensis and reveal the metabolic pathway of root respiration impact the biomass accumulating of G. uralensis. Six groups of one-year-old G. uralensis were fertilized with total nutrition containing various nitrogen concentration (0, 0.5, 1, 2, 4, 8 mmol x L(-1)) every week. At the end of every month, from June to October, the volume respiration rate and biomass of different classes of root samples were determined, and the correlation between root respiration and biomass was analyzed. The results indicated a negative correlation between volume respiration rate and biomass, nitrogen supply significantly affected both root respiration and biomass of G. uralensis by reducing root respiration and increasing root biomass. Under 8 mmol x L(-1) nitrogen supplying, there existed the optimal inhibition of root respiration, which has increased biomass of G. uralensis.

[Effect of Tongfeng trace elements nutrient balance agent on growth, physiological characteristics and content of active constituents of Glycyrrhiza uralensis].

OBJECTIVE: To investigate the effects of Tongfeng trace elements nutrient balance agent on the various growth indicators, physiological indicators, and the contents of liquiritin and glycyrrhizic acid in one-year old Glycyrrhiza uralensis. METHOD: The plants of G. uralensis growing in Chifeng of Inner Mongolia and medicinal garden of Beijing University of Chinese Medicine were fertilized for two times, respectively. The photosynthetic physiological indicators were measured by LI-6400 photosynthetic instrument. The pigments and antioxidase activities of the leaves were determined. Then contents of liquiritin and glycyrrhizic acid in the plants were determined by HPLC. RESULT: The application of this trace element nutrient balance agent could significantly improve the height, chla and chlb, and the photosynthetic physiology indicator such as P(n), C(i), and G(s). Similarly, it could significantly increase the fresh weight of shoots and dry weight of the roots. Compared with control block (CK), the fertilizer which was diluted by 300 times (T(1)) and 600 times (T(2)) significantly increased the content of glycyrrhizic acid by 24.72% and 20. 23%. There was significant difference between different treatments (P < 0.05). CONCLUSION: The Tongfeng trace elements nutrient balance agent could promote growth, physiology and the content of active constituents of G. uralensis, especially the effect of T(1) was superior to T(2).

[Study on the inducement and influential factors of callus with different organs of Glycyrrhiza uralensis].

OBJECTIVE: To investigate the influential factors and establish culture method for G. uralensis callus. METHODS: To study the possible effective factors of culture condition by comparing with different explants, light, plant hormones and its ratio. RESULTS: The hypocotyl was the best among different explants, its inducing ratios was 94%, and the callus occurred earliest. When the calluses were inoculated on MS + 6 - BA (1.0-2.0) mg/L + NAA (0.5-1.0) mg/L, adventitious buds formed. 2,4-D could induce non-embryogenic callus, the compounding proportions of 6 - BA and NAA could induce embryogenic callus. Light influenced induction and growth of callus. CONCLUSION: Different explants, components of hormone and light are the influencial factors of callus induction and growth of G. uralensis.

[Effects of carbon source and nitrogen source on callus growth and flavonoid content in Glycyrrhiza uralensis].

OBJECTIVE: To study the effects of carbon source and nitrogen source on callus growth, and flavonoid content in Glycyrrhiza uralensis. METHOD: Induction and culture of callus were conducted in the media of different concentrations of sucrose, and fructose and ratio of NH4+/NO3- ,and flavonoid content was measured by HPLC. RESULT: The results showed that fructose was superior to sucrose for callus growth and flavonoid formation, and the optimum concentration was 2%. The flavonoids content was 2 times higher than that of sucrose as carbon sources. In the range of 2% -6% of fructose concentration, the flavonoid content was decreased along with the concentration of fructose, but the licochalcone was increased 5-fold. The highest flavonoid content of 151.47 microg x g(-1) was obtained when the ratio of NH4+/NO3- in the medium was 1/2. NH; inhibited the callus growth and flavonoid formation of G. uralensis. CONCLUSION: Fructose as carbon source was superior to sucrose for callus growth and flavonoid formation, and NO3- was favorable to the callus growth and flavonoids accumulation.