The Ammonium/Nitrate Ratio Affects the Growth and Shikonin Accumulation in Arnebia euchroma

Abstract

Nitrogen (N) strongly affects plant growth and metabolism. Although ammonium toxicity has been reported, the effects of nitrogen on shikonin biosynthesis remain obscure. In this study, we tested four different concentrations of NH₄⁺ on Arnebia euchroma hairy roots (AEHR) to clarify the influence of NH₄⁺ on the growth of AEHR and on shikonin accumulation in them and the possible mechanisms. The results showed that compared with the 0% NH₄⁺ treatment (only nitrate as a nitrogen source), the 10% NH₄⁺ treatment increased the fresh weight and the dry weight of AEHR and promoted the synthesis of shikonins. In contrast, the 20% NH₄⁺ treatment started to show inhibition effects on the growth of and shikonin accumulation in AEHR, and the 30% NH₄⁺ treatment exhibited the strongest inhibition effects. With an increased percentage of NH₄⁺, the AEHR became shorter and thicker, with more branches. To further elucidate the mechanisms, we analyzed the time course of nitrogen assimilation, the gene expression level of key enzymes involved in shikonin biosynthesis pathway, and the content of various endogenous hormones in the presence of toxic NH₄⁺ concentrations. The results indicated that auxin and cytokinin might regulate the growth and architecture of AEHR under NH₄⁺ toxicity and revealed that the jasmonate level was reduced along with the inhibition of shikonin biosynthesis. This first comprehensive investigation of the effects of the ammonium/nitrate ratio on shikonin biosynthesis not only provides valuable data for optimizing the in vitro culture of A. euchroma and its shikonin production, but also suggests potential fertilizing strategies for its cultivation.

1. Introduction

Arnebia euchroma (Royle) Johnst., a species of Boraginaceae Arnebia, is one of the important resources of the traditional Chinese medicine Arnebiae Radix. Its major bioactive molecules are shikonin and its derivatives, a class of compounds in most Boraginaceae plants, which exert anti-inflammatory [1], antiviral [2], antioxidant [3], and anti-tumor [4] activities and have an inhibitory effect on topoisomerase I [5]. Shikonin and its derivatives are red naphthoquinone pigments, which can be used in textile dyeing and in the food and cosmetic industry, having a high commercial value [6,7,8]. Tissue culture technology was used to produce shikonin since the 1970s, initially from Lithospermum erythrorhizon [9]. Compared to L. erythrorhizon, A. euchroma is a better source of shikonin-related compounds, harboring red naphthoquinone pigments [10]. While wild A. euchroma has become endangered because of overexploitation, its artificial cultivation is still immature, though the production of shikonin compounds by tissue culture is necessary to meet the market demand.

For the in vitro production of shikonin, White’s medium without NH₄⁺ was firstly applied in a suspension culture of L. erythrorhizon [11]. It was reported that when the NH₄⁺ concentration was increased up to 3% and 30% in White’s medium, the synthesis of shikonin in L. erythrorhizon was completely inhibited, and the cell biomass was decreased by more than half. After that, a medium without NH₄⁺ was widely used for shikonin production in vitro using cell, tissue, and organ cultures of L. erythrorhizon, A. euchroma, and Onosma paniculatum. To optimize the culture conditions, the effects of various medium compositions, abiotic/biotic elicitors, and signal transduction molecules on shikonin synthesis in callus, cell suspension, and hairy root cultures have been widely studied [9,11,12,13,14,15,16]. However, the effects of nitrogen on shikonin synthesis remain elusive. In addition, no mechanistic research on NH₄⁺-dependent inhibition of shikonin synthesis has been published.

Nitrogen is a primary nutrient essential for plant growth. Plants can absorb two types of inorganic nitrogen from the soil: nitrate nitrogen (NO₃⁻-N) and ammonium nitrogen (NH₄⁺-N). Both the absolute nitrogen amount and the ratio of ammonium to nitrate in the soil can change the nitrogen metabolism in plants. The nitrogen-containing organic compounds may affect many physiological and metabolic plant activities by affecting the synthesis of many structural and functional bioactive molecules, which is reflected in the growth, development, and also secondary metabolism of plants. It was generally found that the nitrogen metabolism of crops was most active when providing a mixed ammonium nitrate nutrition, and an appropriate nitrogen administration can significantly promote crop growth and effective compounds accumulation. For example, the nitrate reductase (NR) and glutamine synthetase (GS) activities in Poncirus trifoliata (L.) Raf [17] seedling root were the highest in the presence of a NH₄⁺/NO₃⁻ ratio of 50:50, and the total amounts of organic acids, total alkaloids, and adenosine in Pinellia pedatisecta was the highest at a ratio of NH₄⁺/NO₃⁻ of 75:25 [18].

In addition to being involved in nitrogen metabolism, NO₃⁻ can also act as a signaling molecule to regulate many physiological processes, with a significant regulatory effect on the expression of genes related to other metabolic pathways [19,20]. Although some studies have found that NH₄⁺-N may also be a signaling molecule, other studies have verified that excessive NH₄⁺-N can produce significant toxic effects in plants in the following ways: (i) the absorption of NH₄⁺-N will cause environmental acidification and inhibit the absorption of other cations; (ii) a large amount of free NH₄⁺ will destroy the transmembrane proton gradient and affect cell metabolism; (iii) ineffective assimilation to avoid free NH₄⁺ toxicity will consume a large amount of energy and carbohydrates, breaking the carbon and nitrogen metabolic balance and affecting respiration and photosynthesis; (iv) by causing hormone metabolism imbalance, etc.

Wild A. euchroma grows in alpine meadow soil rich in humus. We collected the inter-root soil of A. euchroma and measured its content of inorganic nitrogen NH₄⁺-N and NO₃⁻-N. We found that it contained 17.384 mg/kg of NH₄⁺-N and 44.486 mg/kg of NO₃⁻-N (unpublished results). The natural high ratio of NH₄⁺ (28%) led us to explore what NH₄⁺ concentration was favorable for the growth of and shikonin accumulation in A. euchroma.

In this study, we investigated the effects of NH₄⁺ on the growth of and shikonin synthesis in A. euchroma hairy roots (AEHR), as well as the possible mechanisms of great importance for shikonin production in in vitro cultures of A. euchroma. Based on our findings, we also investigated a fertilizer strategy for farming. In fact, as the regulation of root-specific metabolites can be mimicked in hairy root cultures as an experimental model system, the results from this study can also direct fertilizer strategies for A. euchroma farming.

2. Materials and Methods

2.1. Materials and Regents

A. euchroma seeds for aseptic seedling were collected in Xinjiang, China. Agrobacterium Rhizogenes (Strain C58C1) was provided by the Resource Center of Chinese Materia Medica, China Academy of Chinese Medical Sciences. AEHR were inducted in the C58C1-infected cotyledons of sterile plantlets of A. euchroma as described previously [8]. AEHR were cultured in MS ammonia-free liquid medium (50 mL) for extended culture.

Both acetonitrile and methanol were LC-grade and were supplied by Merck Company. A Water Purification System from Milli-Q (Millipore, Bedford, MA, USA) was used to acquire ultrapure water. Information regarding the standards is shown in Supplemental data (Table S1).

2.2. Medium Formulation

Using MS medium, AEHR were treated with four nutrient solutions with different NH₄⁺ concentrations, i.e., 0% NH₄⁺, 10% NH₄⁺, 20% NH₄⁺, and 30% NH₄⁺, under the premise of controlling that the total inorganic nitrogen was 20 mM. To avoid large differences for other major elements in the nutrient solutions with different NH₄⁺ concentrations, the concentrations of major elements in MS medium were optimized (Table S2). The amounts of trace elements, iron salts, and organic components in each nutrient solution containing a different NH₄⁺ concentration were the same as in MS medium. All media were adjusted to pH = 5.80.

2.3. Experimental Design

Long-term treatment for 15 d

About 0.05 g of AEHR at the same growth status cultured in ammonia-free MS solid medium for 11 days were separately transferred to 20 mL of different media with 0%, 10%, 20%, and 30% NH₄⁺. AEHR treated for 10 d, 13 d, and 15 d were then analyzed for the determination of their fresh weight, dry weight, and content of shikonin compounds. AEHR treated for 15 d were also used for the determination of root morphological indexes.

Short-term treatment for 48 h

AEHR (0.2 g) at the same growth status cultured in ammonia-free MS solid medium for 11days were transferred to ammonia-free MS liquid medium (20 mL). After 8 days, the original medium was removed, and we added 20 mL of the different media containing 0%, 10%, 20%, and 30% NH₄⁺. The AEHR were treated with different concentrations of NH₄⁺ for 0 h, 6 h, 12 h, 24 h, 36 h, and 48 h and then harvested. One-half of AEHR was used to determine the hormone content (fresh), a quarter of AEHR was used to determine the expression level of key enzyme genes possibly involved in shikonin biosynthesis (fresh), and the rest of AEHR was used to determine the content of shikonin compounds. The short-term treatment was repeated, and the AEHR (0.10 g) were precisely weighed for the determination of their nitrate nitrogen content and ammonia nitrogen content (fresh).

2.4. Mass Weight Measurement and Root Morphology Analysis

We eliminated the excess medium on the AEHR surface with absorbent paper, determined the fresh weight and dry weight at 40 °C until reaching a constant weight, and calculated the drying rate = (dry weight/fresh weight) × 100%. Then, we carefully separated the AEHR with tweezers, collected images under the root scanner, and calculated the number of root branches, root length, root diameter, root surface area, and root volume with the WinRHIZO software (Regant, Quebec, QC, Canada). Three biological replicates were performed for each treatment, and three complete AEHR w from each biological replicate were used to obtain the root morphological indexes.

2.5. Quantification of Shikonin Compounds

UPLC was carried out with a Waters Acquity UPLC-PDA system equipped with a Waters HSS T3 column (2.1 mm × 100 mm, 1.8 μm), measuring the absorbance at 516 nm. The column temperature was set at 40 °C. For shikonin content determination in the hairy roots, about 20 mg of lyophilized hairy root was extracted by ultrasonication in 1 mL of methanol. The mobile phase comprised acetonitrile (A) and water (0.1% formic acid, B) and was introduced at 0.5 mL min⁻¹ with the following gradient program (0–2.0 min, 10.0–55.0% A; 2.0–2.5 min, 55.0–59.0% A; 2.5–7.0 min, 59.0–65.0% A; 7.0–8.0 min, 65.0–65.6% A; 8.0–14.0 min, 65.6–79.0% A; 14.0–14.1 min, 79.0–98.0% A; 14.1–16.0 min, 98.0–98.0% A; 16.0–16.1 min, 98.0–10.0% A; 16.1–18.0 min, 10–10% A).

2.6. Determination of NO₃-N and NH₂-N Contents

The content of NH₂-N was determined by using the Plant Ammonia Nitrogen Assay Kit (Youxuan BIC, Shanghai, China) based on the reaction of ammonia with hypochlorite and phenol in alkaline conditions, which produces the blue-colored indophenol. NO₃-N was measured with the Plant Nitrate Nitrogen Assay Kit (Youxuan BIC, Shanghai, China), which is based on the reaction of NO₃-N with salicylic acid to produce the yellow nitrosalicylic acid under alkaline conditions (pH > 12). The absorbance changes were measured by a microplate reader (Bio-Rad, Kyoto, Japan).

2.7. RNA Extraction and qRT-PCR Analysis

Total RNA was extracted from AEHR using the GK reagent (Huayueyang, Beijing, China), following the manufacturer’s instructions. After treatment with DNAses, the samples were fractionated on an agarose gel to analyze RNA integrity and genomic DNA contamination. The first-strand cDNAs were synthesized with the Primer Script First Strand cDNA Synthesis Kit with random primers and oligo (dT) (TaKaRa, Dalian, China). qRT-PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) and an Applied Biosystems 7500 real-time instrument [21]. We chose the enzymes of AePAL, AeHMGR, AePGT, and CYP76B74 in this study. AeHMGR catalyzes a key step in the upstream mevalonate pathway of shikonin biosynthesis, and AePAL participates in the upstream phenylpropanoid pathway. AePGT, the connection of these two pathways, catalyzes the formation of an important precursor to shikonin. CYP76B74 catalyzes the formation of 3″-hydroxy-geranylhydroquinone, and its gene is the first one involved in the downstream pathway of shikonin synthesis in A. euchroma that was discovered and cloned (Figure S1) [8]. The primers used are listed in Supplemental Table S3. The 18S rRNA was used as an endogenous control to normalize the expression data [22]. At least three independent experiments were performed for each analysis.

2.8. Sample Preparation for Hormone Determination

Powder samples ground in liquid nitrogen ere extracted with an isopropanol/hydrochloric acid (10 mL) extraction buffer; then, of 1 µg/mL of an internal standard solution (8 µL) was added, and the samples were shaken at 4 °C for 30 min. Then, 20 mL of dichloromethane was added to the samples, which were shaken at 4 °C for another 30 min. After centrifuging at 13,000 r/min for 5 min at 4 °C, the lower organic phase was separated and dried with nitrogen away from the light. We then redissolved it with 400 µL of methanol (0.1% formic acid), filtered the solution by a 0.22 µm filter membrane, and analyzed it by UPLC-MS/MS.

2.9. Quantification of Hormones in AEHR

UPLC-MS/MS was carried out with a QTRAP 6500 mass spectrometer (ABSCIEX), connected with an Acquity UPLC system (Waters). The UPLC system was equipped with poroshell 120 SB-C18 (2.1 mm × 150 mm, 2.7 μm) reversed-phase column. The column temperature was set at 40 °C. The mobile phase comprised methanol (A) and water (0.1% formic acid, B) and was introduced at 0.5 mL min⁻¹ with the following gradient program (0–1.0 min, 20% A; 1.0–9.0 min, 20–80% A; 9.0–10.0 min, 80% A; 10.0–10.1 min, 80–20% A; 10.1–15.0 min, 20% A). The mass spectrometric parameters were as follows curtain gas, 15 psi; spray voltage, 4500 V; atomizer pressure, 65 psi; auxiliary gas pressure, 70 psi; atomization temperature, 400 °C.

2.10. Statistical Analysis

The statistical analysis of the obtained results was performed by One-Way ANOVA using the LSD test at a significance level of p < 0.05. All statistical tests were performed using SPSS 22.0 (Chicago, IL, USA).

3. Results

3.1. Growth of AEHR in Response to Ammonium

The medium used in this study was MS medium with two types of inorganic nitrogen, i.e., NH₄⁺ and NO₃⁻ (Table S2). When A. euchroma hairy roots (AEHR) were cultured in media with a consistent total nitrogen content corresponding to 20 mM but different percentages of NH₄⁺ (0%, 10%, 20%, 30%), the AEHR showed obvious differences in root characteristics including surface color (representing the content of shikonin, Figure 1).

AEHRs were harvested after being cultured for 10 d, 13 d, and 15 d. The fresh weight and dry weight of each sample were measured (Figure 2). The amounts of biomass significantly increased from 0 d to 15 d, and the trend of the changes varied with the NH₄⁺ concentrations. Compared to 0% NH₄⁺, 10% NH₄⁺ highly increased both the fresh weight (by 1.58–1.97-fold) and the dry weight (by 1.19–1.67-fold) of AEHR after culturing for 10 days. In addition, 20% NH₄⁺ also significantly increased the fresh weight of AEHR, but this effect was relatively weaker than that observed with 10% NH₄⁺. There was only a promotion of dry matter accumulation after 10 d. When the NH₄⁺ concentration increased to 30%, we observed a strong inhibition effect on dry matter accumulation. A significant decrease in dry weight at 13 d and 15 d was observed, even though there was a slight increase in fresh weight. It was interesting to notice that the drying rates with 10% NH₄⁺, 20% NH₄⁺, and 30% NH₄⁺ were significantly lower than that with 0% NH₄⁺, in spite of the effect on biomass accumulation. Overall, with an increasing ammonium concentration, the growth of AEHR was first promoted (especially with 10% NH₄⁺) and then inhibited (with 30% NH₄⁺).

3.2. Accumulation of Shikonin in AEHR in Response to Ammonium

The red color of the roots could roughly be due to the accumulation of shikonin and its derivatives. It was observed that the roots treated with 10%, 20%, and 30% NH₄⁺ turned red at 10 d, 13 d, and 15 d, respectively, after transfer to fresh medium (Figure 1), while in the 0% NH₄⁺ condition, the AEHR remained red at 5 d and 8 d. These results indicated that the NH₄⁺ concentration affected the accumulation and content of shikonin. The contents of six main shikonin compounds in AEHR treated with different concentrations of NH₄⁺ for 10 d, 13 d and 15 d were quantified by UPLC (Figure 3). After 10 days of treatment, except for deoxyshikonin (DS), the concentration of five shikonin derivatives in AEHR under the 10% NH₄⁺ treatment was notably less than without NH₄⁺ (0% NH₄⁺) and close to zero or not detected in the presence of 20% NH₄⁺ and 30% NH₄⁺. However, the concentrations of DS, the precursor of other shikonin compounds, in 10% NH₄⁺ and 0% NH₄⁺ were comparable. After culturing for 13 days, the AEHR under the 10% NH₄⁺ treatment showed the highest concentrations of β-hydroxyisovalerylshikonin, acetylshikonin, and β,β′-dimethacrylicalkannin (HIVS, AS, and DAA), which were, respectively, 18.33%, 77.34%, and 71.99% higher than those in 0% NH₄⁺ and 162.85%, 80.99%, and 82.54% higher than those in 20% NH₄⁺. AEHR treated with 20% NH₄⁺ began to accumulate shikonin components, especially DS, while the accumulation of all kinds of shikonin derivatives in 30% NH₄⁺ was still strongly suppressed, except for a visible increase in DS.

When continuing the treatments for 15 days, both the 10% NH₄⁺ and the 20% NH₄⁺ treatments promoted the synthesis of shikonin compounds in the AEHR compared with the 0% NH₄⁺ treatment, except for DS. When considering the cumulative effects on the six shikonin derivatives, the 10% NH₄⁺ treatment had the strongest promotion effect, allowing to obtain a dry weight of 8.59 mg/g, which was 2.17-fold higher than that obtained with the 0% NH₄⁺ treatment. The dry weight obtained with 20% NH₄⁺ was also significantly higher than that obtained with 0% NH₄⁺ (p < 0.05), i.e., 0.90-fold higher than that achieved with 0% NH₄⁺ (with 20% NH₄⁺, it reached 5.16 mg/g). For roots under the 30% NH₄⁺ treatment, the dry weight remained very low (less than 1 mg/g) even after 15 d, which indicated a fierce inhibition of shikonin biosynthesis.

3.3. Development and Morphology of AEHR in Reponse to Ammonium

The WinRHIZO root-scanning system was used to investigate the morphological characters of hairy roots harvested at 15 d (Figure 4a). The AEHR treated with 0% NH₄⁺ had the longest length (99.73 cm) and the smallest diameter (0.28 mm). With the increase of NH₄⁺ concentration, the AEHR gradually became shorter and thicker (Figure 4b,c). This was also indicated by the length of roots with a diameter in the range from 0.00 mm to 0.25 mm (L1) and the percentage of total root length (PTL). These roots were 71.57 ± 5.62 cm, 44.20 ± 2.59, 32.60 ± 7.26, and 25.68 ± 9.04 cm in length (L1) and accounted for 71.76%, 59.82%, 47.05%, and 37.12% of PTL, respectively, under the conditions of 0% NH₄⁺, 10% NH₄⁺, 20% NH₄⁺, and 30% NH₄⁺. For thicker roots, with a diameter in the range between 0.25 and 0.50 mm (L2), 0.50 and 1.00 mm (L3), and 1.00 and 1.50 mm (L4), the PTL at different NH₄⁺ concentrations showed almost the same trend, which was 0% NH₄⁺ < 10% NH₄⁺ < 20% NH₄⁺ < 30% NH₄⁺ (Figure 4h,i).

Although the root length varied, the root surface area (SA) and root projection area (PA) of the AEHR showed no significant difference under different NH₄⁺ treatments. In addition, the root volume (V) increased with the increase of NH₄⁺ concentration and was significantly higher under the 30% NH₄⁺ treatment than in the presence of 0% NH₄⁺ (p < 0.05). The branch number (B) also increased with the increase in NH₄⁺ concentration, and the number of AEHR branches at 20% NH₄⁺ was significantly higher than that at 0% NH₄⁺ (p < 0.05, Figure 4d–g).

3.4. Nitrogen Assimilation in AEHR under Short-Term Ammonium Treatments

In general, the NO₃-N and NH₂-N content in plants is directly related to the level of NO₃-N and NH₄⁺-N in the environment, respectively. Since NO₃⁻ absorption consumes more energy than NH₄⁺ absorption, plants tend to absorb NH₄⁺. Studies have shown that the presence of NH₄⁺ inhibited NO₃⁻ absorption [23,24]. Meanwhile, in order to avoid free-ammonium toxicity, they rapidly transform NH₄⁺-N into NH₂-N. In order to investigate the possible mechanism by which NH₄⁺ concentration affected AEHR growth and shikonin compounds production, we conducted short-term ammonium treatments for 48 h. The contents of NO₃-N and NH₂-N in AEHR within 48 h after treatment with different concentrations of NH₄⁺ were measured (Figure 5). Under the 0% NH₄⁺ treatment for 48 h, both NO₃-N and NH₂-N contents in AEHR fluctuated slightly, which indicated that nitrogen metabolism was relatively balanced. Compared with the 0% NH₄⁺ treatment, the other three (10%, 20%, and 30% NH₄⁺) treatments inhibited NO₃-N accumulation in AEHR and increased NH₂-N content in AEHR. Under the 10% NH₄⁺ treatment, the content of NH₂-N in the AEHR increased sharply within 12 h and then remained stable, with a slight decline. For the 20% NH₄⁺ and 30% NH₄⁺ treatments, the content of NH₂-N increased sharply for 24 h and then maintained a stable high level of around 4000 μg/g, which might cause the normal nitrogen metabolism and ammonium toxicity to inhibit the growth of and shikonin synthesis in AEHR.

3.5. Shikonin Biosynthesis in AEHR under Short-Term Ammonium Treatments

The contents of six main shikonin derivatives in the AEHR under short-term treatments at different NH₄⁺ concentrations were measured by UPLC (Figure 6). Compared with AEHR at 0 h (before being transferred to a fresh medium), the contents of shikonin derivatives in the AEHR decreased at all NH₄⁺ concentrations. This decrease was likely because the hairy roots required time to adapt to the fresh culture medium and preferred a nutrient-dependent growth rather than secondary production at the early stage of the growth period. Another reason could be that the shikonin derivatives were secreted at the root surface and dispersed into the fluid medium. Compared with the 0% NH₄⁺ treatment, the 10% NH₄⁺ treatment led to significantly increased contents of HIVS, AOIVA, and IBS at 36 h and of HIVS and IBS at 48 h (p < 0.05). Meanwhile, the inhibition effects of the 20% NH₄⁺ and 30% NH₄⁺ treatments were not that obvious, except for DS and AS, whose contents displayed a significant decrease (p < 0.05) compared to those at 0% NH₄⁺. Compared to the contents obtained with the 10% NH₄⁺ treatment carried out for 48 h, the contents of all the six shikonin derivatives decreased with the 20% NH₄⁺ and 30% NH₄⁺ treatments, and there were significant differences in the HIVS, IBS, DS, and AS levels (p < 0.05).

3.6. Gene Expression of Key Enzymes in the Shikonin Biosynthesis Pathway

The biosynthesis pathway of shikonin has been well dissected [8,25,26]. AeHMGR, AePAL, AePGT, and CYP76B74 were proven to be genes for key enzymes regulating the accumulation of shikonin, in previous studies [8,26]. To reveal the effect of NH₄⁺ on shikonin biosynthesis at the gene expression level, their time-dependent expression levels after treatment with the different NH₄⁺ concentrations were quantified with qRT-PCR (Figure 7). Compared with the 0% NH₄⁺ treatment, 10% NH₄⁺ increased the expression levels of AeHMGR, AePAL, AePGT, and CYP76B74 after 6 h of treatment, while the relative expression levels of the AeHMGR, AePAL, and AePGT were downregulated by the 20% NH₄⁺ and 30% NH₄⁺ treatments. After treating for 36 h, their expression levels appeared significantly different. The expression levels of AeHMGR, AePAL, AePGT, and CYP76B74 after treatment with 10% NH₄⁺ were upregulated about 1.36-fold, 0.79-fold, 1.05-fold, and 0.78-fold, respectively, compared to those at 0% NH₄⁺. These genes after treatment with 30% NH₄⁺ were downregulated 0.84-fold, 0.67-fold, 0.71-fold, and 0.29-fold, respectively. The 20% NH₄⁺ treatment also led to a downregulated gene expression of AeHMGR, AePAL, AePGT, and CYP76B74 compared to the 0% NH₄⁺ treatment, but to higher gene expression levels of AeHMGR, AePAL, AePGT (but not CYP76B74) than the 30% NH₄⁺ treatment. The consistent results of gene expression and metabolite accumulation under different NH₄⁺ concentrations indicated that 10% and 30% NH₄⁺ influenced the accumulation of shikonin compounds by regulating the expression of genes involved in shikonin biosynthesis pathway.

3.7. Hormone Metabolism of AEHR under NH₄⁺ Toxicity

NH₄⁺ toxicity is common in plants, but the NH₄⁺ threshold after which it appears varies between species [27]. Studies indicated that NH₄⁺ toxicity is associated with hormonal disruption in plants [28]. As AE is a plant sensitive to NH₄⁺ toxicity, the levels of eight hormones present in AEHR treated with 0% NH₄⁺ and 30% NH₄⁺ for 48 h were determined in this study, to profile the possible hormonal disruption caused by NH₄⁺ toxicity in AEHR (Figure 8). Compared with 0% NH₄⁺, the 30% NH₄⁺ treatment promoted the synthesis of the endogenous cytokinins ZT and TZR and of auxin IAA, but not of IPA. The 30% NH₄⁺ treatment had a stronger effect on cytokinin than on auxin, the content of ZT was significantly higher than with 0% NH₄⁺ at 12 h, 36 h, and 48 h (p < 0.05), and the content of TZR was extremely significantly higher (p < 0.01). For endogenous auxin, there was no significant difference, and consistent results were obtained for the 0% NH₄⁺ and the 30% NH₄⁺ treatments during 48 h. The increased level of cytokinin might be responsible for the shorter root length and the thicker diameter of AEHR under the 30% NH₄⁺ treatment.

Previous studies demonstrated that different exogenous hormones have various effects on shikonin accumulation. For instance, GA [29] and ABA have significantly negative impacts on shikonin biosynthesis, while MeJA and SA [12,16,30,31] can effectively promote it. In our study, the content of endogenous GA₄ in AEHR under the 30% NH₄⁺ treatment did not change significantly within 48 h, suggesting that GA may not be involved in the regulation of NH₄⁺ toxicity in AEHR. As for ABA, its content after the 30% NH₄⁺ treatment decreased rapidly and remained at a low and steady level for 24 h, as observed with the 0% NH₄⁺ treatment, which might be related to the adaptation of the hairy roots to the new medium. Then, it began to rise after treatment for 36 h and reached the high level of 0.11 ng/g at 48 h. This was in contrast to the trend of shikonin accumulation and the expression of key enzymes involved in the shikonin biosynthesis pathway, indicating ABA as a negative regulator of the shikonin biosynthesis pathway.

The content of endogenous MeJA grew for 24 h after treatment with 0% and 30% NH₄⁺, then in the latter conditions, it decreased and, in the former conditions (0% NH₄⁺), it reached the highest level at 36 h before decreasing. The trend of SA levels under treatment with 0% NH₄⁺ was similar to that of MeJA; it increased for 36 h then decreased, while it decreased slowly in the first 24 h and then somewhat fluctuated with 30% NH₄⁺. The content of SA in AERH treated with 0% NH₄⁺ at 12 h, 24 h, and 36 h was significantly (p < 0.01) higher than that in AERH treated with 30% NH₄⁺. These results are consistent with the tendency of shikonin accumulation and relative gene expression. The inhibition by high concentrations of NH₄⁺ of shikonin production was likely associated with the decrease in endogenous MeJA and SA, especially SA.

4. Conclusions

In conclusion, A. euchroma is an ammonium-sensitive species, and we found that 10% NH₄⁺ (2 mM) was beneficial for both the growth of AEHR and the accumulation of shikonin compounds compared to ammonium as the sole nitrogen source (0% NH₄⁺), while 20% NH₄⁺ and 30% NH₄⁺ had strong inhibitory effects. With an increased percentage of NH₄⁺, the AEHRs became shorter and thicker, with more branches. Additional experiments to elucidate the mechanism underlying these observations indicated that under NH₄⁺ toxicity, auxin and cytokinin may regulate the growth and architecture of AEHR. Furthermore, the biosynthesis of shikonin was inhibited, along with the production of MeJA and SA. This comprehensive investigation into the effects of the ammonium/nitrate ratio on shikonin biosynthesis provides valuable data for optimizing the in vitro culture of A. euchroma and its shikonin production and suggests potential fertilizing strategies for its cultivation.

5. Discussion

5.1. Arnebiae Euchroma Is an Ammonium-Sensitive Species

Nitrogen is an essential element for the growth and development of plants. It was discovered in 1882 that ammonium can be toxic to plants, leading to a variety of issues such as stunted rhizomes, shortened roots, yellowing leaves, and even death. Different species and varieties of plants have varying levels of tolerance to ammonium. In this study, when the concentration of ammonium was raised to 20% NH₄⁺ (4 mM), inhibition began to occur. Both the growth of AEHR and shikonin production could be strongly inhibited by 30% NH₄⁺ (6 mM). These results demonstrated that A. euchroma is an ammonium-sensitive plant, exhibiting similar ammonium sensitivity to other ammonium-sensitive species which would display ammonium toxicity when exposed to ammonium concentrations of less than 10 mM, such as Arabidopsis, citrus, wheat, and L. erythrorhizon. Arabidopsis is a highly ammonium-sensitive plant. In fact, 1 mM ammonium was found to impede the growth of the taproot, as well as reduce the expansion of cells and cell yield [32]. Furthermore, ammonium was also found to inhibit the geotropism of roots. Citrus plants exposed to a high ammonium concentration (8 mM) exhibited growth inhibition, metabolic disorder, decreased biomass, abnormal morphology, and a reduced growth rate [33]. As for wheat, the plant biomass, total root length, surface area, and root volume were significantly reduced by 5 mM ammonium [34]. Cell cultures of L. erythrorhizon, another shikonin-producing species closely related to A. euchroma, were reported to be ammonium-sensitive. When NH₄⁺ concentration was increased up to 3% and 30% in White’s medium, the synthesis of shikonin was completely inhibited, and the cell biomass decreased by more than half, respectively. Similar results were obtained in the present study, as both the growth and shikonin production of AEHR increased as the NH₄⁺ proportion increased to 20% and 30%, but the plant was not as extremely sensitive as L. erythrorhizon. Differently from ammonium-sensitive plants, Spartina alterniflora is highly tolerant to ammonium, which can even alleviate salt stress [35,36]. Additionally, ammonium nitrogen has been found to be more beneficial for the growth of and nutrient accumulation in Pinus massoniana (Masson’s pine) tissue culture seedlings than nitrate nitrogen. Therefore, it is recommended to apply an ammonium nitrogen fertilizer when cultivating Masson’s pines seedlings. The preference of different species may be influenced by the ammonium+/nitrate ratio found in their native habitats, and optimization studies can facilitate the development of fertilization schedules for their cultivation.

5.2. Compare to a Sole Nitrate Nitrogen Source, A. euchroma Prefers an Appropriate Ammonium/Nitrate Ratio

For most plants, a mixed nitrate and ammonium nitrogen source is superior to ammonium or nitrate nitrogen sources alone in terms of plant growth and chemical components accumulation [34,37,38]. The optimal ratio of ammonium/nitrate for plant growth depends on plant species, environmental conditions, plant developmental stage, and total concentration of supplied nitrogen [39]. Ammonium nitrogen can promote the growth of the aboveground parts of the coffee plant, but an excessive concentration of it (ammonium/nitrate ratio above 70:30) can decrease the chlorophyll content and inhibit the growth of the aboveground parts. On the other hand, an increase in nitrate nitrogen is beneficial for the growth of roots and the underground parts but can have an adverse effect on the growth of leaves, stems, and other aboveground parts. As a result, a 50:50 ammonium/nitrate ratio leads to a higher biomass yield of coffee seedlings [40]. In the present study, it was clarified that 10% NH₄⁺ (2 mM) was beneficial for both the growth of AEHR and the accumulation of shikonin compounds compared to ammonium as the sole nitrogen source (0% NH₄⁺), even though A. euchroma is an ammonium-sensitive plant.

We reviewed all the media used for the in vitro culture of A. erchroma, L. erythorhizon, and O. paniculatum, and the concentrations of NH₄⁺ and NO₃⁻, the total content of nitrogen, and the NH₄⁺/NO₃⁻ ratio were compared (Table 1). A two-step method is widely used for the in vitro culture of L. erythorhizon, i.e., biomass is first accumulated in growth medium, and then a shikonin-production medium is used for shikonin accumulation. That is because of the different nutrients necessary for the two stages of L. erythorhizon cultures, especially nitrogen. For the in vitro culture of A. erchroma, studies were mostly performed using a one-step method. The total content of nitrogen varied from around 10 mM to 60 mM, and the NH₄⁺/NO₃⁻ ratio from 0% to 34.34%, and most studies used medium without NH4⁺ to avoid ammonium toxicity. In present study, the optimum NH₄⁺/NO₃⁻ ratio for both growth and shikonin production was 10%. We highly recommended the B5 and SH media for the in vitro culture of A. erchroma, especially for the hairy roots.

5.3. Auxin and Cytokinin Might Regulate the Growth and Architecture of AEHR under NH₄⁺ Toxicity

Studies demonstrated that NO₃⁻ and NH₄⁺ are involved in hormone regulation during plant root formation and leaf development. Compared with NO₃⁻, NH₄⁺ is more likely to cause an imbalance in plant hormone metabolism, which is one of the mechanisms of ammonium toxicity. Under NH₄⁺ toxicity, we found that both growth and architecture changed. Compared with AERH in the presence of the sole nitrate nitrogen source, AEHR after the 30% NH₄⁺ treatment were shorter and thicker, with more branches. These changes were linked to alterations in hormonal balance. We speculated that they were linked to increased auxin and cytokinin production in the roots. Under ammonium toxicity, AEHR in this study showed significant changes in both endogenous auxin (IAA, IPA) and cytokinin (ZT, TZR) levels. Ammonium toxicity increased IPA content at 6 h, and IAA, ZT, and TZR contents at 12 h, 36 h, and 48 h. This is consistent with the results of Yang et al. for Arabidopsis thaliana cultured using a single ammonium nitrogen source and based on matrix transcriptomes [54]. In the taproot grown in (NH₄)₂SO₄, the nutrient and metabolic imbalance induced by ammonium was partially overcome by the increase in the auxin level. Dziewit et al. [55] studied the temporal and spatial distribution of auxin in Arabidopsis thaliana under long-term ammonium toxicity and found that auxin accumulated in the leaves and roots, but not in the root tips. The apparent auxin pattern in different tissues is associated with the developmental adaptation of ammonium-growing plants with short branches and highly branched roots. Ammonium toxicity is known to affect auxin activity, which serves as an important positive regulator of lateral root (LR) development [56]. In studies on general NH₄⁺ toxicity, it was suggested that more prolific root branching resulted from the increased strength of the root tissue acting as a carbon sink under NH₄⁺ nutrition, which would facilitate auxin delivery to the roots. The increased number of root tips, which has been often observed, could then lead to the increased production of cytokinins in ammonium-grown plants. The increased ZT and TZR contents at 12 h, 36 h, and 48 h are in accord with this theory. A study of NH₄⁺ toxicity alleviation by Si also suggested a dependence on the increase in the trans-ZT content in the shoots. In cytokinin-deficient plants, Si did not alleviate NH₄⁺ toxicity [57]. However, in some studies, the cytokinin showed an opposite performance. Walch-Liu et al. reported that NH₄⁺ inhibition of plant growth was associated with a sharp decline in cytokinin concentration [58].

5.4. Shikonin Synthesis in AEHR under NH₄⁺ Toxicity and Its Possible Hormonal Regulation Mechanism

Plant hormones, especially MeJA and SA, play an important role in regulating plant secondary metabolism and environmental responses. Apart from their effects on growth, those on shikonin biosynthesis regulation are various, depending on the different forms of auxin or cytokinin and the plant species. It was reported that IAA could positively regulate shikonin accumulation in L. erythorhizon, while IBA inhibited it in A. euchroma cells. Synthetic auxin 2,4-D and NAA exerted consistent negative regulatory effects on shikonin biosynthesis in cells of L. erythorhizon, A. euchroma, and O. paniculatum [59,60]. Among the cytokinins, KT and 6-BA promoted shikonin biosynthesis in the L. erythorhizon callus but inhibited it in A. euchroma cells [59,61].

Studies have shown a consistent promotional effect of shikonin biosynthesis in L. erythorhizon, A. euchroma [21], L. officinale [62], and O. paniculatum [63]. Exogenous MeJA promoted the accumulation of shikonin derivatives, shikonofuran derivatives, and rosmarinic acid in shikonin-proficiency cells and caused a rapid and massive increase in the expression of genes involved in the shikonin biosynthesis pathway, such as HMGR and PGT [21]. Zhao et al. [16,31] overexpressed LeMYB1 in the hairy roots of L. erythorhizon. The content of shikonin compounds increased significantly, and at the same time, the expression of LeMYB1 showed the same positive response to JA. Additionally, the endogenous JA content was significantly increased when the L. erythorhizon cells were transferred from the B5 medium (for the growth step) to the M9 medium (for the shikonin production step) [64]. In contrast, the responses to exogenous SA were not as consistent as those to JA. Kumar et al. reported that exogenous SA can inhibit the synthesis of shikonin in A. euchroma cells, i.e., 10 µM SA reduced by nearly 80% shikonin production, and 100 µM SA completely inhibited shikonin production. They found the addition of exogenous SA caused a decreased activity of PHB geranyltransferase, one of the key regulatory enzymes in shikonin biosynthesis [30]. In another study, Arghavani et al. reported higher enhancing effects of SA compared to JA [12]. There is no report on the relationship between the endogenous content of SA and shikonin production. It is clear that the SA could improve the enzymatic activity of PAL in the phenylpropane pathway [30], where p-hydroxybenzoic acid, one of the precursors of shikonin, is produced. In our study, the endogenous MeJA and SA levels were decreased by the 30%NH₄⁺ treatment for 48 h, which might be related to the inhibition of shikonin biosynthesis.

Author Contributions

Conceptualization, S.W. and L.G.; sample preparation and measurement, L.S., J.L., B.Y. and C.L.; data analysis, L.S., J.L., X.W., Q.L. and M.C.; writing—original draft preparation, L.S. and J.L.; writing—review and editing, S.W., R.W. and Y.Z.; supervision, L.G.; funding acquisition, S.W. and R.W. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Chunjuan Pu for her support in using the WinRHIZO.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Figures and Table

Figure 1. Continuous growth of AEHR in 15 days after treatment with different concentrations of NH4+ (0%, 10%, 20%, 30%).

Figure 2. Biomass differences in AEHR under different concentrations of NH4+. (a) The fresh weight of AEHR. (b) The dry weight of AEHR. (c) The drying rate of AEHR.The values shown are means ± SE (n = 3 replicates). Significant differences (p < 0.05) are indicated by different letters for each variable.

Figure 3. Effects of different concentrations of NH4+ on the synthesis of shikonin during 15 days. The vertical bars represent the mean ± SE (n = 3). (a) HIVS, β-hydroxyisovalerylshikonin; (b) AS, acetylshikonin; (c) DS, deoxyshikonin; (d) AOIVA, β-acetoxyisovalerylalkannin; (e) IBS, isobutyrylshikonin; (f) DAA, β,β′-dimethacrylicalkannin.

Figure 4. Morphological characters of AEHR after treatment for 15 days with different NH4+ concentrations. (a) Morphological characters of AEHR after treated for 15 days with different NH4+ concentrations; (b,c) average length and average diameter of AEHR; (d) oot surface area; (e) root projected area; (f) mean root volume; (g) branch number; (h,i) AEHR length (L) for different diameters and percentage of total root length (PTL). Different letters represent significant differences (n = 3, mean ± SE, p < 0.05).

Figure 5. Contents of NO3-N and NH2-N in AERH at different NH4+ concentrations during 48 h. (a) The concent of NH2-N in AEHR. (b) The concent of NO3-N in AEHR. Vertical bars represent the mean ± SE (n = 3).

Figure 6. Effects of different concentrations of NH4+ on the synthesis of shikonin during 48 h. Vertical bars represent the mean ± SE (n = 3). (a) HIVS, β-hydroxyisovalerylshikonin; (b) AS, acetylshikonin; (c) DS, deoxyshikonin; (d) AOIVA, β-acetoxyisovalerylalkannin; (e) IBS, isobutyrylshikonin; (f) DAA, β,β′-dimethacrylicalkannin.

Figure 7. Effects of different concentrations of NH4+ on the gene expression of key enzymes in the shikonin biosynthesis pathway during 36 h. (a) The gene expression of CYP76B74. (b) The gene expression of AeHMGR. (c) The gene expression of AePGT. (d) The gene expression of AePAL. Vertical bars represent the mean ± SE (n = 3).

Figure 8. Impact of the 30% NH4+ treatment on auxin and cytokinin levels in AEHR during 48 h. (a) IAA, Auxin; (b) IPA, Indolepropionic acid; (c) ZT, Zeatin; (d) TZR, trans-Zeatin-riboside; (e) GA4, Gibberellin4; (f) ABA, Abscisic acid; (g) MeJA, Methyl Jasmonate; (h) SA, Salicylic Acid. Vertical bars represent the mean ± SE (n = 3).

Table 1

Media used for the in vitro culture of A. erchroma, L. erythorhizon, and Onosma paniculatum retrieved from the literature.

Varieties	Step	Medium	NH4+ (mM)	NO3− (mM)	Total Content (mM)	NH4+/NO3− Ratio	References
L. erythorhizonsuspensioncell cultures	Two-step for growth	LS	20.61	39.4	60.01	34.34%	[11]
		MG-5	6.25	54.22	60.47	10.34%	[6]
	Two-step for shikonin production	White	0	3.33	3.33	0.00%	[11]
		M9	0	6.67	6.67	0.00%	[11]
L. erythorhizon hairy roots	Two-step for growth	B5	2.02	24.73	26.75	7.55%	[16,31]
	Two-step for shikonin production	M9	0	6.67	6.67	0.00%	[16,31]
A. erchroma suspension cell cultures	One-step	AG-7	7	18.79	25.79	27.14%	[41,42]
	Two-step for growth	AG-7	7	18.79	25.79	27.14%	[42]
		LS	20.61	39.4	60.01	34.34%	[43]
		MS	20.61	39.4	60.01	34.34%	[44,45]
	Two-step for shikonin production	M9	0	6.67	6.67	0.00%	[42,46,47,48,49,50,51]
		M10	0	10.97	10.97	0.00%	[43]
		APM	0	17.74	17.74	0.00%	[44,45]
A. erchroma hairy roots	Two-step for growth	SH	2.61	24.73	27.34	9.55%	[51]
		SH without NH4+	0	24.73	24.73	0.00%	[47,50,52]
		MS without NH4+	0	18.79	18.79	0.00%	[47,52]
		B5 without NH4+	0	24.73	24.73	0.00%	[47]
		1/2MS without NH4+	0	9.395	9.395	0.00%	[46,52]
		LS without NH4+	0	18.79	18.79	0.00%	[47]
		MG-5 without NH4+	0	47.97	47.97	0.00%	[52]
		MS	20.61	39.4	60.01	34.34%	[47]
		B5	2.02	24.73	26.75	7.55%	[49]
	Two-step for shikonin production	M9	0	6.67	6.67	0.00%	[47,50]
Onosma paniculatum suspension cell cultures	Two-step for growth	B5	2.02	24.73	26.75	7.55%	[53]
	Two-step for shikonin production	M9	0	6.67	6.67	0.00%	[53]

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13051318/s1, Table S1: Information on the standards used for quantification; Table S2: Primers for qRT-PCR; Table S3: Mass element formulation of media with different NH₄⁺ concentration (mM). Figure S1: Role of enzymes in the shikonin biosynthetic pathways.

References

1. Shindo, S.; Hosokawa, Y.; Hosokawa, I.; Ozaki, K.; Matsuo, T. Shikonin Inhibits Inflammatory Cytokine Production in Human Periodontal Ligament Cells. Inflammation; 2016; 39, pp. 1124-1129. [DOI: https://dx.doi.org/10.1007/s10753-016-0344-0] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27072015]

2. Cocchi, F.; DeVico, A.L.; Yarchoan, R.; Redfield, R.; Cleghorn, F.; Blattner, W.A.; Garzino-Demo, A.; Colombini-Hatch, S.; Margolis, D.; Gallo, R.C. Higher macrophage inflammatory protein (MIP)-1alpha and MIP-1beta levels from CD8+ T cells are associated with asymptomatic HIV-1 infection. Proc. Natl. Acad. Sci. USA; 2000; 97, pp. 13812-13817. [DOI: https://dx.doi.org/10.1073/pnas.240469997] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11095721]

3. Tong, Y.; Bai, L.; Gong, R.; Chuan, J.; Duan, X.; Zhu, Y. Shikonin Protects PC12 Cells Against β-amyloid Peptide-Induced Cell Injury Through Antioxidant and Antiapoptotic Activities. Sci. Rep.; 2018; 8, 26. [DOI: https://dx.doi.org/10.1038/s41598-017-18058-7] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29311595]

4. Wang, B.; Wu, Z.; Wang, J.; Li, W.; Liu, G.; Zhang, B.; Tang, Y. Insights into the mechanism of Arnebia euchroma on leukemia via network pharmacology approach. BMC Complement. Med. Ther.; 2020; 20, 322. [DOI: https://dx.doi.org/10.1186/s12906-020-03106-z] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33109189]

5. Ahn, B.Z.; Baik, K.U.; Kweon, G.R.; Lim, K.; Hwang, B.D. Acylshikonin analogues: Synthesis and inhibition of DNA topoisomerase-I. J. Med. Chem.; 1995; 38, pp. 1044-1047. [DOI: https://dx.doi.org/10.1021/jm00006a025]

6. Yazaki, K. Lithospermum erythrorhizon cell cultures: Present and future aspects. Plant Biotechnol.; 2017; 34, pp. 131-142. [DOI: https://dx.doi.org/10.5511/plantbiotechnology.17.0823a]

7. Papageorgiou, V.P.; Assimopoulou, A.N.; Couladouros, E.A.; Hepworth, D.; Nicolaou, K.C. The Chemistry and Biology of Alkannin, Shikonin, and Related Naphthazarin Natural Products. Angew. Chem. Int. Ed. Engl.; 1999; 38, pp. 270-301. [DOI: https://dx.doi.org/10.1002/(SICI)1521-3773(19990201)38:3<270::AID-ANIE270>3.0.CO;2-0]

8. Wang, S.; Wang, R.; Liu, T.; Lv, C.; Liang, J.; Kang, C.; Zhou, L.; Guo, J.; Cui, G.; Zhang, Y. et al. CYP76B74 Catalyzes the 3″-Hydroxylation of Geranylhydroquinone in Shikonin Biosynthesis. Plant Physiol.; 2019; 179, pp. 402-414. [DOI: https://dx.doi.org/10.1104/pp.18.01056]

9. Tabata, M.; Mizukami, H.; Hiraoka, N.; Konoshima, M. Pigment formation in callus cultures of Lithospermum erythrorhizon. Phytochemistry; 1974; 13, pp. 927-932. [DOI: https://dx.doi.org/10.1016/S0031-9422(00)91425-0]

10. Fu, S.L.; Shang, T.M.; Xiao, P.G. Analysis of naphthoquinone pigments in several Arnebiae Radix. Acta Pharm. Sin.; 1984; 19, pp. 921-925. [DOI: https://dx.doi.org/10.16438/j.0513-4870.1984.12.008]

11. Fujita, Y.; Hara, Y.; Ogino, T.; Suga, C. Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon: I. Effects of nitrogen sources on the production of shikonin derivatives. Plant Cell Rep.; 1981; 1, pp. 59-60. [DOI: https://dx.doi.org/10.1007/BF00269272] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24258859]

12. Arghavani, P.; Haghbeen, K.; Mousavi, A. Enhancement of Shikalkin Production in Arnebia euchroma Callus by a Fungal Elicitor, Rhizoctonia solani. Iran. J. Biotechnol.; 2015; 13, pp. 10-16. [DOI: https://dx.doi.org/10.15171/ijb.1058] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28959304]

13. Fang, R.; Zou, A.; Zhao, H.; Wu, F.; Zhu, Y.; Zhao, H.; Liao, Y.; Tang, R.J.; Pang, Y.; Yang, R. et al. Transgenic studies reveal the positive role of LeEIL-1 in regulating shikonin biosynthesis in Lithospermum erythrorhizon hairy roots. BMC Plant. Biol.; 2016; 16, 121. [DOI: https://dx.doi.org/10.1186/s12870-016-0812-6] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27230755]

14. Srinivasan, V.; Ryu, D.D. Improvement of shikonin productivity in Lithospermum erythrorhizon cell culture by alternating carbon and nitrogen feeding strategy. Biotechnol. Bioeng.; 1993; 42, pp. 793-799. [DOI: https://dx.doi.org/10.1002/bit.260420702]

15. Tani, M.; Takeda, K.; Yazaki, K.; Tabata, M. Effects of oligogalacturonides on biosynthesis of shikonin in Lithospermum cell cultures. Phytochemistry; 1993; 34, pp. 1285-1290. [DOI: https://dx.doi.org/10.1016/0031-9422(91)80017-U]

16. Zhao, H.; Baloch, S.K.; Kong, L.R.; Zhang, W.J.; Zou, A.L.; Wang, X.M.; Qi, J.L.; Yang, Y.H. Molecular cloning, characterization, and expression analysis of LeMYB1 from Lithospermum erythrorhizon. Biol. Plant.; 2014; 58, pp. 436-444. [DOI: https://dx.doi.org/10.1007/s10535-014-0411-z]

17. Sun, M.H.; Lu, X.P.; Cao, X.J.; Li, J.; Xiong, J.; Xie, X.X. Effect of different forms of nitrogen on the activity of nitrate reductase and expression of the relative genes in Citrus sinensis ×Poncirus trifoliate. J. Fruit Sci.; 2017; 34, pp. 410-417. [DOI: https://dx.doi.org/10.13925/j.cnki.gsxb.20160314]

18. Wang, Q.; Wang, K.C.; Zheng, C.X.; Zhu, B.C.; Wang, Y.L.; Yang, J.W.; Wang, W. Effect of different nitrogenous forms on growth and chemical component in tuber of Pinellia pedatisecta Schott. J. Plant Nutr. Fertil.; 2014; 20, pp. 1038-1043. [DOI: https://dx.doi.org/10.11674/zwyf.2014.0428]

19. Crawford, N.M.; Glass, A.D.M. Molecular and physiological aspects of nitrate uptake in plants. Trends Plant Sci.; 1998; 3, pp. 389-395. [DOI: https://dx.doi.org/10.1016/S1360-1385(98)01311-9]

20. Migocka, M.; Warzybok, A.; Kłobus, G. The genomic organization and transcriptional pattern of genes encoding nitrate transporters 1 (NRT1) in cucumber. Plant Soil; 2012; 364, pp. 245-260. [DOI: https://dx.doi.org/10.1007/s11104-012-1345-x]

21. Wang, S.; Guo, L.P.; Xie, T.; Yang, J.; Tang, J.F.; Li, X.; Wang, X.; Huang, L.Q. Different secondary metabolic responses to MeJA treatment in shikonin-proficient and shikonin-deficient cell lines from Arnebia euchroma (Royle) Johnst. Plant Cell Tissue Organ Cult. PCTOC; 2014; 119, pp. 587-598. [DOI: https://dx.doi.org/10.1007/s11240-014-0558-5]

22. Kuchipudi, S.V.; Tellabati, M.; Nelli, R.K.; White, G.A.; Perez, B.B.; Sebastian, S.; Slomka, M.J.; Brookes, S.M.; Brown, I.H.; Dunham, S.P. et al. 18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells. Virol. J.; 2012; 9, 230. [DOI: https://dx.doi.org/10.1186/1743-422X-9-230] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23043930]

23. Gazzarrini, S.; Lejay, L.; Gojon, A.; Ninnemann, O.; Frommer, W.B.; von Wirén, N. Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. Plant Cell; 1999; 11, pp. 937-948. [DOI: https://dx.doi.org/10.1105/tpc.11.5.937] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/10330477]

24. Xu, Q.F.; Tsai, C.L.; Tsai, C.Y. Interaction of potassium with the form and amount of nitrogen nutrition on growth and nitrogen uptake of maize. J. Plant Nutr.; 1992; 15, pp. 23-33. [DOI: https://dx.doi.org/10.1080/01904169209364299]

25. Song, W.; Zhuang, Y.; Liu, T. CYP82AR Subfamily Proteins Catalyze C-1′ Hydroxylations of Deoxyshikonin in the Biosynthesis of Shikonin and Alkannin. Org. Lett.; 2021; 23, pp. 2455-2459. [DOI: https://dx.doi.org/10.1021/acs.orglett.1c00360] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33728922]

26. Yazaki, K.; Kunihisa, M.; Fujisaki, T.; Sato, F. Geranyl diphosphate:4-hydroxybenzoate geranyltransferase from Lithospermum erythrorhizon. Cloning and characterization of a ket enzyme in shikonin biosynthesis. J. Biol. Chem.; 2002; 277, pp. 6240-6246. [DOI: https://dx.doi.org/10.1074/jbc.M106387200] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/11744717]

27. Britto, D.T.; Kronzucker, H.J. NH4+ toxicity in higher plants: A critical review. J. Plant Physiol.; 2002; 159, pp. 567-584. [DOI: https://dx.doi.org/10.1078/0176-1617-0774]

28. Esteban, R.; Ariz, I.; Cruz, C.; Moran, J.F. Review: Mechanisms of ammonium toxicity and the quest for tolerance. Plant Sci. Int. J. Exp. Plant Biol.; 2016; 248, pp. 92-101. [DOI: https://dx.doi.org/10.1016/j.plantsci.2016.04.008]

29. Yoshikawa, N.; Fukui, H.; Tabata, M. Effect of gibberellin A₃ on shikonin production in Lithospermum callus cultures. Phytochemistry; 1986; 25, pp. 621-622. [DOI: https://dx.doi.org/10.1016/0031-9422(86)88011-6]

30. Kumar, P.; Saini, M.; Bhushan, S.; Warghat, A.R.; Pal, T.; Malhotra, N.; Sood, A. Effect of salicylic acid on the activity of PAL and PHB geranyltransferase and shikonin derivatives production in cell suspension cultures of Arnebia euchroma (Royle) Johnst—A medicinally important plant species. Appl. Biochem. Biotechnol.; 2014; 173, pp. 248-258. [DOI: https://dx.doi.org/10.1007/s12010-014-0838-x]

31. Zhao, H.; Chang, Q.S.; Zhang, D.X.; Fang, R.J.; Zhao, H.; Wu, F.Y.; Wang, X.M.; Lu, G.H.; Qi, J.L.; Yang, Y.H. Overexpression of LeMYB1 enhances shikonin formation by up-regulating key shikonin biosynthesis-related genes in Lithospermum erythrorhizon. Biol. Plant.; 2015; 59, pp. 429-435. [DOI: https://dx.doi.org/10.1007/s10535-015-0512-3]

32. Liu, Y.; Lai, N.; Gao, K.; Chen, F.; Yuan, L.; Mi, G. Ammonium inhibits primary root growth by reducing the length of meristem and elongation zone and decreasing elemental expansion rate in the root apex in Arabidopsis thaliana. PLoS ONE; 2013; 8, e61031. [DOI: https://dx.doi.org/10.1371/journal.pone.0061031]

33. Chen, H.; Jia, Y.; Xu, H.; Wang, Y.; Zhou, Y.; Huang, Z.; Yang, L.; Li, Y.; Chen, L.-S.; Guo, J. Ammonium nutrition inhibits plant growth and nitrogen uptake in citrus seedlings. Sci. Hortic.; 2020; 272, 109526. [DOI: https://dx.doi.org/10.1016/j.scienta.2020.109526]

34. Hachiya, T.; Watanabe, C.K.; Fujimoto, M.; Ishikawa, T.; Takahara, K.; Kawai-Yamada, M.; Uchimiya, H.; Uesono, Y.; Terashima, I.; Noguchi, K. Nitrate Addition Alleviates Ammonium Toxicity Without Lessening Ammonium Accumulation, Organic Acid Depletion and Inorganic Cation Depletion in Arabidopsis thaliana Shoots. Plant Cell Physiol.; 2012; 53, pp. 577-591. [DOI: https://dx.doi.org/10.1093/pcp/pcs012]

35. Hessini, K.; Kronzucker, H.J.; Abdelly, C.; Cruz, C. Drought stress obliterates the preference for ammonium as an N source in the C₄ plant Spartina alterniflora. J. Plant Physiol.; 2017; 213, pp. 98-107. [DOI: https://dx.doi.org/10.1016/j.jplph.2017.03.003]

36. Hill, T.D.; Sommer, N.R.; Kanaskie, C.R.; Santos, E.A.; Oczkowski, A.J. Nitrogen uptake and allocation estimates for Spartina alterniflora and Distichlis spicata. J. Exp. Mar. Biol. Ecol.; 2018; 21, pp. 466-472. [DOI: https://dx.doi.org/10.1016/j.jembe.2018.07.006]

37. Garnica, M.; Houdusse, F.; Zamarreno, A.M.; Garcia-Mina, J.M. The signal effect of nitrate supply enhances active forms of cytokinins and indole acetic content and reduces abscisic acid in wheat plants grown with ammonium. J. Plant Physiol.; 2010; 167, pp. 1264-1272. [DOI: https://dx.doi.org/10.1016/j.jplph.2010.04.013]

38. Petersen, F.; Demann, J.; Restemeyer, D.; Ulbrich, A.; Olfs, H.W.; Westendarp, H.; Appenroth, K.J. Influence of the Nitrate-N to Ammonium-N Ratio on Relative Growth Rate and Crude Protein Content in the Duckweeds Lemna minor and Wolffiella hyalina. Plants; 2021; 10, 1741. [DOI: https://dx.doi.org/10.3390/plants10081741]

39. Mantovani, C.; Prado, R.M.; Pivetta, K.F.L. Impact of Nitrate and Ammonium ratio on Nutrition and Growth of two Epiphytic Orchids. An. Acad. Bras. Cienc.; 2018; 90, pp. 3423-3431. [DOI: https://dx.doi.org/10.1590/0001-3765201820171008]

40. Carr, N.F.; Boaretto, R.M.; Mattos, D., Jr. Coffee seedlings growth under varied NO₃⁻:NH₄⁺ ratio: Consequences for nitrogen metabolism, amino acids profile, and regulation of plasma membrane H⁺-ATPase. Plant Physiol. Biochem.; 2020; 154, pp. 11-20. [DOI: https://dx.doi.org/10.1016/j.plaphy.2020.04.042]

41. Hao, H.; Lei, C.; Dong, Q.; Shen, Y.; Chi, J.; Ye, H.; Wang, H. Effects of exogenous methyl jasmonate on the biosynthesis of shikonin derivatives in callus tissues of Arnebia euchroma. Appl. Biochem. Biotechnol.; 2014; 173, pp. 2198-2210. [DOI: https://dx.doi.org/10.1007/s12010-014-1025-9] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24974168]

42. Kang, Q.S.; Li, H.L.; Wu, Y.L.; Lu, D.Y. Studies on cell growth and Product Synthesis in Production Stage Culture of Arnebia euchroma Cell II: Fermentation culture. Nat. Prod. Res. Dev.; 2003; 15, pp. 429-432. [DOI: https://dx.doi.org/10.16333/j.1001-6880.2003.05.015]

43. Ge, F.; Yuan, X.; Wang, X.; Zhao, B.; Wang, Y. Cell growth and shikonin production of Arnebia euchroma in a periodically submerged airlift bioreactor. Biotechnol. Lett.; 2006; 28, pp. 525-529. [DOI: https://dx.doi.org/10.1007/s10529-006-0014-7]

44. Malik, S.; Bhushan, S.; Sharma, M.; Ahuja, P.S. Physico-chemical factors influencing the shikonin derivatives production in cell suspension cultures of Arnebia euchroma (Royle) Johnston, a medicinally important plant species. Cell Biol. Int.; 2011; 35, pp. 153-158. [DOI: https://dx.doi.org/10.1042/CBI20090459] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/20977433]

45. Malik, S.; Bhushan, S.; Verma, S.C.; Sharma, N.; Sinha, A.K.; Sharma, M.; Ahuja, P.S. Production of naphthoquinone pigments in cell suspension cultures of Arnebia euchroma (Royle) Johnston: Influence of pH on growth kinetics and acetylshikonin. Med. Aromat. Plant Sci. Biotechnol.; 2008; 2, pp. 43-49.

46. Liang, J.W.; Li, T.; Wang, R.S.; Zhou, L.; Yang, Q.; Wang, S.; Guo, L.P. Establishment of RNA interfered hairy root system of two CYP450 genes in Arnebia euchroma and its influence. China J. Chin. Mater. Med.; 2020; 45, pp. 3422-3431. [DOI: https://dx.doi.org/10.19540/j.cnki.cjcmm.20200506.110]

47. Lu, W.H.; Chen, Y.F.; Wang, F.; Dai, N.B.; Hao, A.H.; Li, C.F.; Jia, S.E. Effects of physical and chemical conditions on hairy root culture and content of Arnebia euchromu. J. Huazhong Agric. Univ.; 2012; 31, pp. 50-54. [DOI: https://dx.doi.org/10.13300/j.cnki.hnlkxb.2012.01.016]

48. Zhang, P.; Wang, F.; Zhu, C. Influence of fungal elicitor and macroporous resin on shikonin accumulation in hairy roots of Arnebia euchroma (Royle) Johnst. Chin. J. Biotechnol.; 2013; 29, pp. 214-223. [DOI: https://dx.doi.org/10.13345/j.cjb.2013.02.006]

49. Yang, Y.H.; Lu, J.; Zhao, Q.H.; Cao, R.Q. Effect of water extract of algae on growth and pigment formation in Lithospermuml erythrorhizon and Arnebia euchromu cell cultures. J. Plant Resour. Environ.; 1992; 1, pp. 39-44.

50. Ge, S.N.; Zhang, S.X.; Wang, F.; Wei, H.; Xie, W.L.; Xie, J.; Li, C.; Zhao, H. Effects of Exogenous Substances on the Content of Secondary Metabolites in Arnebia euchroma Johnst Hairy Roots. Food Sci.; 2016; 37, pp. 160-164. [DOI: https://dx.doi.org/10.7506/spkx1002-6630-201609030]

51. Li, M.; Pan, Q.; Wang, F.; Zhang, P. Research of Effects on Arnebia euchroma (Royle) Johnst Hairy Roots Growth Factors. Xinjiang Agric. Sci.; 2012; 49, pp. 2062-2068. [DOI: https://dx.doi.org/10.6048/j.issn.1001-4330.2012.11.016]

52. Lu, W.H.; Pan, Q.; Wang, F.; Chen, Y.F.; Dai, N.B. Influence of Culture Conditions on Arnebia euchromaHairy Roots Growth and Shikonin Content. Acta Bot. Boreali Occident. Sin.; 2012; 32, pp. 1686-1691. [DOI: https://dx.doi.org/10.3969/j.issn.1000-4025.2012.08.027]

53. Yang, Y.; Zhang, H.; Cao, R. Effect of Brassinolide on Growth and Shikonin Formation in Cultured Onosma paniculatum Cells. J. Plant Growth Regul.; 1999; 18, pp. 89-92. [DOI: https://dx.doi.org/10.1007/PL00007054]

54. Yang, H.; von der Fecht-Bartenbach, J.; Friml, J.; Lohmann, J.U.; Neuh User, B.; Ludewig, U. Auxin-modulated root growth inhibition in Arabidopsis thaliana seedlings with ammonium as the sole nitrogen source. Funct. Plant Biol.; 2015; 42, pp. 239-251. [DOI: https://dx.doi.org/10.1071/FP14171]

55. Dziewit, K.; Pěnčík, A.; Dobrzyńska, K.; Novák, O.; Szal, B.; Podgórska, A. Spatiotemporal auxin distribution in Arabidopsis tissues is regulated by anabolic and catabolic reactions under long-term ammonium stress. BMC Plant Biol.; 2021; 21, 602. [DOI: https://dx.doi.org/10.1186/s12870-021-03385-9]

56. Péret, B.; De Rybel, B.; Casimiro, I.; Benková, E.; Swarup, R.; Laplaze, L.; Beeckman, T.; Bennett, M.J. Arabidopsis lateral root development: An emerging story. Trends Plant Sci.; 2009; 14, pp. 399-408. [DOI: https://dx.doi.org/10.1016/j.tplants.2009.05.002]

57. Barreto, R.F.; de Mello Prado, R.; Lúcio, J.C.B.; López-Díaz, I.; Carrera, E.; Carvalho, R.F. Ammonium Toxicity Alleviation by Silicon is Dependent on Cytokinins in Tomato cv. Micro-Tom. J. Plant Growth Regul.; 2021; 41, pp. 417-428. [DOI: https://dx.doi.org/10.1007/s00344-021-10314-5]

58. Walch-Liu, P.; Neumann, G.; Bangerth, F.; Engels, C. Rapid effects of nitrogen form on leaf morphogenesis in tobacco. J. Exp. Bot.; 2000; 51, pp. 227-237. [DOI: https://dx.doi.org/10.1093/jexbot/51.343.227]

59. Fang, D.Q.; Hou, C.S.; Li, X.M.; Ye, H.C.; Li, G.F. Effects of pH and hormone on cell growth and synthesis of Shikonin derivatives in suspension culture of Arnebia euchroma. Plant Sci. J.; 1994; 2, pp. 159-164.

60. Zhu, W.H.; Fan, H.X.; Hu, Q.; Zhu, H.Q. Induction culture of callus and selection of superior clones of Onosma paniculatum. J. Chin. Med. Mater.; 1990; 1, pp. 6-9. [DOI: https://dx.doi.org/10.13863/j.issn1001-4454.1990.01.002]

61. Yan, H.Y.; Cao, R.Q. Influencing factors of Shikonin formation in callus culture of Lithospermum erythorhizon. Acta Agric. Boreali Sin.; 2002; 17, pp. 116-120. [DOI: https://dx.doi.org/10.3321/j.issn:1000-7091.2002.02.025]

62. Ahmad, M.; Alonso, A.V.; Koletti, A.E.; Rodić, N.; Reichelt, M.; Rödel, P.; Assimopoulou, A.N.; Paun, O.; Declerck, S.; Schneider, C. et al. Dynamics of alkannin/shikonin biosynthesis in response to jasmonate and salicylic acid in Lithospermum officinale. Sci. Rep.; 2022; 12, 17093. [DOI: https://dx.doi.org/10.1038/s41598-022-21322-0]

63. Ding, J.; Shi, S.; Jiang, B.-H.; Yang, Y.-H.; Huang, J.; Shen, H.-G.; Xia, K.; Zhang, J.; Jiang, X. Effects of Methyl jasmonate with indole-3-acetic acid and 6-benzylaminopurine on the secondary metabolism of cultured Onosma paniculatum cells. Vitr. Cell. Dev. Biol. Plant; 2004; 40, pp. 581-585. [DOI: https://dx.doi.org/10.1079/IVP2004578]

64. Zhu, Y. Cloning, Expression and Functional Analysis of LeTCP4 Gene in Lithospermum Erythrorhizon. Master’s Thesis; Nanjing University: Nanjing, China, 2012.