Impact of Artificial Polyploidization in Ajuga reptans on Content of Selected Biologically Active Glycosides and Phytoecdysone

Abstract

Polyploidization in plants, which involves doubling or further multiplying of genome, has the potential to improve the constituents that make medicinal plants, like Ajuga reptans, attractive to the pharmaceutical, cosmetics, and food production industries; botanical pesticide effects could also be derived. The aim of this study was to determine how artificial polyploidization in A. reptans plants affected the composition and quantity of biologically active substances from the glycoside and phytoecdysone families. Diploids and artificial tetraploids of A. reptans were analyzed. Changes in the contents of trans-teupolioside, trans-verbascoside, and 20-hydroxyecdysone were evident in the aboveground parts of the cultivated plants (e.g., leaves and flowers). The tetraploid lines of Ajuga plants displayed variability in, and increased levels of, trans-teupolioside and trans-verbascoside content. The 20-hydroxecdysone content was slightly higher in tetraploids. These findings indicated that Ajuga tetraploids could be used in breeding programs to enhance the yield of substances with potential medicinal and industrial applications.

1. Introduction

Artificial polyploidization in plants is a breeding technique used to modify the genome, mainly by doubling (and occasionally further multiplying) chromosome sets. However, because this biotechnological approach affects the genome, it is not classified as genetic modification. Polyploidization techniques are experiencing a renaissance; they are increasingly being incorporated into the breeding programs of many interesting crops and aromatic and medicinal plants [1,2,3,4].

Especially in the cases of aromatic and medicinal plants, polyploidization affects the quantities of a wide range of biologically active substances [3]. For secondary metabolites, in particular, these changes could allow the use of plant extracts in the pharmaceutical, cosmetics, and food processing industries, and as botanical pesticides [5]. One such medicinal plant, which is known for its biologically active substances, is Ajuga reptans (blue bugle), belonging to the family Lamiaceae. Plants from the genus Ajuga have a wide variety of biologically active substances, such as phytoecdysteroids, anthocyanins, carotenoids, diterpenes, and phenylethanoid glycosides. Extracts from this plant species contain two glycosides: teupolioside and verbascoside. Several studies have demonstrated the potential anticancer, anti-inflammatory, and antioxidant effects of this species, which could, therefore, have cosmeceutical and food production applications [6,7]. Moreover, the presence of phytoecdysteroids (20-hydroxyecdysone) suggests the possible use of Ajuga extract as a botanical pesticide. Phytoecdysteroids have the same structural features as the ecdysteroids found in insects and other arthropods. It is believed that plant ecdysteroids have antifeedant effects on herbivores and suppress insect molting [8,9].

Although some researchers have attempted to obtain the abovementioned substances from in vitro or cell suspension cultures [7,10], breeding programs could provide new genotypes for field production and biomass harvesting. This study aimed to address how artificial tetraploids of Ajuga reptans obtained from previous experiments differ from wild progenitor plants, in terms of the contents of three important substances: trans-teupolioside, trans-verbascoside, and 20-hydroxyecdysone.

2. Materials and Methods

2.1. Plant Material

The plants of two diploid progenitor strains of A. reptans (denoted as 4 and 7), originating from the Crop Protection section of the Crop Research Institute (CRI), Prague-Ruzyně, Czech Republic, were used in this study. Tetraploid plants of three strains of clones (sc4, sc28, and sc12) were also analyzed. Tetraploid strains sc4 and sc28 were derived from progenitor strain 7, and sc12 (as well as sc5, for which only the flowers and leaves were analyzed) was derived from strain 4, to represent the lines of viable plants with the characteristic morphological and cytological features of tetraploids, as reported in Švécarová et al. (2018) [11]. The plants of all of these strains were cultivated in field condition, harvested at the maximum flowering stage, and dried at room temperature under enhanced air circulation. Plants growing in field condition were also morphologically characterized. The surface areas of dried, prepared and scanned leaves were measured using Image J software (https://imagej.nih.gov/ij/index.html (accessed on 23 June 2022). Only two pairs of leaves, located exactly under inflorescence, were taken for analysis. Two datasets, one for diploid leaf areas and one for tetraploid leaf areas, were statistically evaluated by t-test (GraphPad software, San Diego, CA, USA).

2.2. Extraction of the Plant Materials

The dried above-ground plant materials were powdered and extracted with methanol for 2 h at room temperature. Three parallel samples were prepared from each plant material. The extracts were stored at −18 °C. Methanol LiChrosolv, gradient grade for LC, was purchased from Merck (Prague, Czech Republic).

2.3. HPLC and LC/MS Analyses

The extracts were analyzed using HPLC (Hewlett Packard 1050), column Phenomenex Luna C18(2) (150 × 2 mm and 3 µm), diode array detector (DAD Agilent G1315B) and fluorescence detector (FLD Agilent G1321A).

Mobile phase A: 5% acetonitrile + 0.1% of o-phosphoric acid; mobile phase B: 80% acetonitrile + 0.1% of o-phosphoric acid. The gradient: 0. min 0% B, 10. min 10% B, 20. min 30% B, 25. min 40% B, 30. min 60% B. The flow rate was 0.25 mL/min, the column temperature was 35° C. The injection volume was 5 µL.

The substances were also analyzed by LC-MS (APCI-LC-MS, LCQ Accela Fleet), with the same column, but, instead of o-phosphoric acid, formic acid was used. Complete identification was performed by LC-NMR.

Quantification was performed by HPLC using calibration curves for 20-hydroxy-ecdysone and trans-verbascoside (syn. acteoside); trans-teupoloside was quantified according to the calibration curve for verbascoside.

Acetonitrile for LC/MS was purchased from Merck (Prague, Czech Republic), o-phosporic acid p.a. from Fluka (Prague, Czech Republic), formic acid from Sigma-Aldrich (Prague, Czech Republic) and standard of trans-verbascoside and 20-hydroxy-ecdysone from Sigma-Aldrich (Prague, Czech Republic).

Datasets of each measured substance for each pair (progenitor diploid strain and its derived tetraploid strain) were statistically evaluated by unpaired t-test (p < 0.01) (GraphPad software).

2.4. NMR Analysis-Identification of trans-Verbascoside and trans-Teupolioside

For the preparation of trans-verbascoside and trans-teupolioside, a commercial HPLC system (Dionex UltiMate 3000, Thermo Fisher Scientific) with a 4.6 × 250 mm HPLC column (Luna C18 (2), Phenomenex, 5 µm, 100 Å pore size) was employed. The separation was performed by isocratic elution using acetonitrile-deuterium oxide system (22% ACN-78% D2O) and monitored at 254 nm. The flow rate was 0.5 mL/min. The concentrated methanol solution (50 μL) was injected multiple times into the HPLC system and fractions of individual chromatographic peaks were collected. The individual fractions were evaporated to dryness and subsequently dissolved in methanol-d4 for NMR spectroscopy analysis.

The NMR data were acquired using a Varian INOVA 500 MHz spectrometer (Varian Inc., Palo Alto, CA, USA) operating at 499.87 MHz for ¹H and 125.70 MHz for ¹³C. The ¹H, COSY and HSQC spectra were used for structure elucidation of trans-teupolioside, while only ¹H NMR spectrum was recorded for trans-verbascoside. NMR spectra were referenced to the line of the solvent (methanol-d4, δ = 3.31 ppm for 1H and δ = 49.00 ppm for ¹³C), see Supplementary Materials. The identification was based on comparison of NMR data of isolated compounds with available literature [12].

3. Results

Diploid and tetraploid A. reptans plants cultivated in field conditions and originated from in vitro cultures showed typical features for their ploidy levels. Plants from tetraploid strains were characterized by more robust growth of the shoots in comparison with diploid progenitor plants (Figure 1A,B). Moreover, the size of tetraploid flowers and leaves was also bigger than in diploid ones (Figure 1C,D). Measured areas of tetraploid leaves were significantly larger (1.7-fold) than for diploid leaves.

The biochemical analyses focused on whole green and flowering shoots, and especially on leaves and flowers. In all studied strains of A. reptans plants, both diploids and tetraploids were identified in the same spectrum, respectively, in the presence of the analyzed substances of trans-teupolioside, trans-verbascoside and 20-hydroxyecdysone. However, differences in their content were revealed. In terms of the 20-hydroxyecdysone content in whole shoots, the differences between diploid progenitor strains were negligible. The comparison of 20-hydroxyecdysone content in tetraploids and diploids (Figure 2) also showed mostly insignificant differences. Similar results were obtained for analyzed leaves and flowers. Only leaves and flowers of sc4 tetraploids contained a significantly higher amount of 20-hydroxyecdysone in relation to the measured content in the diploid progenitor (Figure 3).

On the other hand, changes were found in the contents of phenylpropanoids. In the case of trans-teupolioside content, there was no significant difference among diploid progenitor strains. In addition, the content of trans-teupolioside in whole shoots showed no significant difference between progenitor strain 4 and its derived tetraploid strain sc12 (Figure 2). A non-significantly higher level of trans-teupolioside was detected in tetraploid flowers and leaves compared with diploids (Figure 3). Moreover, the sc28 and sc4 tetraploid strains had higher and lower trans-teupolioside contents, respectively, than progenitor strain 7 (Figure 2).

The trans-verbascoside content in whole shoots showed differences among tetraploid strains derived from progenitor strain 7; sc4 also differed from diploid progenitor strain 7 (Figure 2). Regarding sc4, it showed a 1.87-fold higher trans-verbascoside content than progenitor strain 7. Furthermore, sc12 tetraploids showed markedly and significantly higher trans-verbascoside content (1.7-fold) than diploid progenitor strain 4 (Figure 2). Tetraploid strain sc4 showed higher content of trans-verbascoside not only in whole shoots, but also separately in flowers and leaves (Figure 3).

4. Discussion

The enlarged shoots and flowers of tetraploid plants grown in greenhouses and field conditions accord with previous findings for A. reptans artificial tetraploids cultivated in vitro [12]. The experimental plants were derived from the tetraploids of in vitro cultures. Enlarged leaves, stems, and flowers in artificially polyploidized plants have been documented for many plant species [3,13,14], as also discussed in the review of Trojak-Goluch et al. (2021) [15].

Artificial polyploidization not only enhances biomass production, but also alters the content of various phytochemicals, including secondary metabolites [16,17,18]. We analyzed trans-teupolioside, trans-verbascoside, and 20-hydroxyecdysone, which were also identified in plant extracts from the genus Ajuga [7,19].

Polyploidization can influence plant metabolism, both qualitatively and quantitatively. Tetraploids of several plant species produce terpenoids [13,17], morphine (in the case of Papaver somniferum) [20], and/or secondary metabolites, such as tetrahydrocannabinol and cannabidiol [21]. In phenylpropanoids, secondary metabolites include teupolioside and verbascoside. Studies of the tetraploids of Solanum commersonii [22] and Solanum bulbocastanum [23] revealed higher phenylpropanoid content in S. commersonii compared with the diploid progenitors, However, the tetraploids of S. bulbocastaneum displayed similar (or lower) phenylpropanoid content to those of their diploid parents.

This study demonstrated differences in phenylpropanoid contents in the tetraploid strain. The phytochemical most affected by polyploidization was trans-verbascoside, the content of which was higher in tetraploids compared with diploid progenitors. Verbascoside exerts numerous biological effects, such as anti-inflammatory effects on the skin, and in the intestines and lungs. It also exerts cytoprotective effects through free radical scavenging, which could be useful for treating neurodegenerative diseases, as reviewed by Alipieva et al. (2014) [24]. The nearly two-fold higher trans-verbascoside content in tetraploids, compared to the diploid progenitors, observed in this study indicated that A. reptans could be used in the pharmacological industry. Changes in 20-hydroxyecdysone contents were also examined, and our results suggested that genome doubling affects the expression of genes involved in complex biosynthetic pathways; this applies to both phenylpropanoids and all of the secondary metabolites. New tetraploid genomes are typically unique, and strains with altered secondary metabolite contents can be produced; such genotypes have potential for breeding programs.

5. Conclusions

In this work, it was demonstrated that the method of artificial polyploidization in A. reptans that utilizes in vitro techniques is able not only to double the genome, but also to change the content of bioactive substances in the plant bodies. Here, new tetraploid materials were tested for increases in their phytochemical contents. The phytochemical most increased by polyploidization was trans-verbascoside. Tetraploid plants which are displaying robust features could also represent new genotypes for usage in gardening and landscaping, as a quick ground covering plant for empty areas.