In Vivo Use of 1D and 2D ¹H NMR to Examine the Glycosylation of Scopoletin in Duboisia myoporoides Cell Suspensions^[*]

• Abstract
• Introduction
• Results and Discussion
• Material and Methods
• Cell suspensions of D. myoporoides
• In vivo NMR device
• NMR analysis
• Chemicals
• References

Abstract

Cell suspensions initiated from Duboisia myoporoides–a shrub belonging to the Solanaceae family and being a rich source of tropane alkaloids–previously showed their ability to glycosylate scopoletin into scopolin, which represent coumarins showing health benefits. To investigate the time course of this glycosylation reaction, an in vivo NMR approach was developed using a perfusion system in an 8-mm NMR tube and ¹H NMR with 1D and 2D (TOCSY and NOESY) experiments. The time course of metabolic changes could therefore be followed without any labeling.

Introduction

Carbohydrate transfer reactions occur in all organisms including plants on different organic compounds such as proteins, lipids, saccharides, and secondary metabolites. These metabolic reactions may be glycosylations when the reaction consists in grafting an osidic substituent on a molecule or it may be deglycosylations when the reaction consists of cutting the bond between a molecule and its osidic substituent. These reactions have a wide range of effects including changes in water solubility, chemical stability, chemical reactivity, and biological properties of the molecules.

Glycosylation reactions are provided by enzymes called UDP-dependent (UDP: Uridine-Di-Phosphate) glycosyltransferases (UGTs), which constitute a large family of highly conserved structure enzymes [1]. In Arabidopsis thaliana (L.) Heynh. (Brassicaceae), hundreds of genes encoding these enzymes were identified, and the 3D structure of four UGTs was determined by crystallography. A review of UGT structure states that despite poor sequence conservation between different UGTs, secondary and tertiary structures are highly conserved and predict their substrate specificity [1]. These GTs are called EC 2.4.x.x and belong to a multigene family. A collection of rice and A. thaliana GT clones has been developed for functional genomics studies [2].

Glycosylation processes are often involved in secondary metabolite biosynthesis [3] and lead to the development of biotechnology processes [4]. Alkaloids can exist naturally in glycosylated forms. Glycoalkaloids are commonly described in Solanaceae family plants such as potato and tomato [5]. Glycosylation occurs also in terpene biosynthesis and is of interest to fragrance and aroma development [6]. Phenolic compounds can also be glycosylated, and this reaction has implications on the regulation of their biosynthesis [7].

Among the glycosylation reactions of plant phenolic compounds, the one studied in this paper concerns the glucosylation of scopoletin to scopolin.

Scopoletin is a coumarin ([Fig. 1])–that is, it consists of the benzopyran-2-one skeleton, which is in this case substituted by a methoxy group on carbon 6 and by a hydroxyl group on carbon 7. The first publication on scopoletin is Mooreʼs 1911 article, which describes the production of this molecule from a Solanaceae, Scopolia japonica Maxim. [8]. Since then, more than a thousand works that deal with scopoletin have been published. This molecule is described in many plants of the Solanaceae family; many studies focus on the well-known Nicotiana plants [9], Solanum [10], and other lesser known plants such as Duboisia myoporoides R. Br. (Solanaceae) [11]. Several more recent studies on the plant model A. thaliana also refer to scopoletin [12], [13], [14], [15], [16].

Scopoletin and scopolin belong to coumarins. These molecules are derived from the phenylpropanoid pathway. The precise biosynthesis and its regulation process are still not well defined. It is known that the scopoletin precursor is a ferul-ate/-oylCoA derivative and/or coumar-ate/-oylCoA and/or caffe-ate/-oylCoA derivative and the biosynthesis involves mainly action of a ligase, a hydroxylase and O-methyltransferases. The glucosylation of scopoletin into scopolin occurs through action of the enzyme EC: 2.4.1.128 [17]. The study by Siwinska et al. in 2014 [15], using a quantitative trait loci (QTL) approach, allowed to elucidate, from a genetic and molecular point of view, the scopoletin and scopolin biosynthesis in A. thaliana where these molecules accumulate in roots.

Coumarins can play a role as phytoalexins intervening in the defense mechanisms of the plant during a biotic or abiotic stress [18]. Their production is organ-specific [19]. They thus participate in plant-fungi interactions [20], and for example, the synthesis of scopoletin occurs during an attack by the fungal plant pathogens Alternaria alternata in tobacco [21] or by Fusarium oxysporum in Ipomea [22]. Scopoletin is also involved in the plant defense against attack by mites such as Tetranychus cinnabarinus [23]. These properties for the plant-insecticidal, antibacterial, acaricide, allelopathic, as well as the main pharmacological properties such as antitumoral, hypolipidemic, spasmolytic, and choleretic are described in a 2012 review [24]. Since the publication of this review, there are other works that show the properties of scopoletin as an anti-inflammatory [25] or an antiallergic [26] or the effect of scopoletin on monoamine oxidases and brain amines [27]. As to the properties of scopolin, few articles are devoted exclusively to this topic. Scopolin is most often included, such as scopoletin, in the study of different coumarins or in the study of properties of plants containing many natural substances–including scopolin–with various biological properties. There is, however, an article that discusses the interesting anti-inflammatory properties of scopolin in osteoarthritis in rats [28], another older article that discusses the modulation of intestinal absorption of hyoscyamine by scopolin [29], and another article investigating the accumulation of scopolin in infected potato tissue [30]. To get a better knowledge of the reaction leading to scopolin from scopoletin, we decided to follow the glycosylation reaction of scopoletin into scopolin in real time while performing in vivo NMR experiments. Despite relatively low sensitivity, in vivo NMR is interesting as a noninvasive and nondestructive analytical technique. ¹H, ¹³C, ¹⁵N, and ³¹P are the most frequently used nuclei, the choice depending on the molecule class accumulated in plants. Different plant tissues can be used for such analyses like roots, leaves (in the dark), fruits, seeds, or cell suspensions. For reviews in this field, see the following papers [31], [32], [33], [34], [35].

Results and Discussion

In order to follow the glycosylation reaction of scopoletin into scopolin in real time, we performed in vivo NMR experiments. [Fig. 2] represents the in vivo ¹H spectra of D. myoporoides cells before and after the addition of scopoletin recorded at several times. Before addition of scopoletin (time 0 h), the main peak visible at 4.75 ppm corresponds to the water signal. Many other peaks are visible in the region between 0 and 4 ppm; they correspond to the proton chemical shifts mainly of amino acids and carbohydrates present in the cells. In the region between 6 and 8 ppm, two peaks are visible at 6.88 and at 7.70 ppm. They correspond to the amino protons of glutamine, which is accumulated at a high level in the D. myoporoides suspension cells. In this latter region, after the addition of scopoletin, three peaks appear at 6.34, 7.22, and 7.96 ppm. They agree well respectively with the H3, H5, and H4 peaks of scopoletin ([Table 1]), though a formal distinction with scopolin is impossible given the in vivo peak width. The H8 peak is probably under the Gln peak. The three peaks increase over time and shift upfield. This may correspond to the uptake and accumulation of scopoletin inside cells and/or to its glycosylation.

To try to distinguish scopolin from scopoletin, 2D NMR experiments were performed. [Fig. 3] represents the in vivo TOCSY spectrum of D. myoporoides cells 9 h after the addition of scopoletin. The total, correlation spectroscopy sequence enables us to correlate protons via geminal or vicinal spin couplings and can give a total correlation of all protons of a chain of coupled spin systems with each other. The most prominent spin systems in the spectrum correspond to glutamine (2.17, 2.48, and 3.82 ppm) and to γ-aminobutyric acid (1.92, 2.34, and 3.05 ppm). Sucrose can also be identified from the characteristic H1 at 5.42 ppm (see inset with the H1 – H2 and H1 – H3 cross-peaks), though the mixing time is too short to correlate the whole spin system. In the aromatic region, the cross-peak visible at 6.36/7.92 ppm corresponds to the correlation between H3 and H4 of scopolin or scopoletin. This cross-peak reflects the accumulation time course of scopolin and scopoletin. As visible on the enlargements on the right, it becomes larger over time and shifts upfield, possibly reflecting a chemical or a pH modification. Proton chemical shifts are indeed known to be sensitive to pH [36], and compartmentation in a plant can provide different pH environments. This is notably the case of cytoplasmic and vacuolar pH. In order to investigate this hypothesis, ¹H NMR spectra of scopoletin standards were recorded at different pHs and the chemical shifts of the coumarin protons are reported graphically in [Fig. 4]. It appears that the chemical shifts of all the ¹H of the coumarin moiety are sensitive to pH. They decrease in the region between approximately pH 6.5 and 9.5, and a closer inspection allows us to determine a pK_a of the phenomenon of 7.8. The shift upfield could correspond to the uptake of scopoletin from medium to cytoplasm since the medium was adjusted to pH 5.8 and the cytoplasmic pH is known to be around 7.5 [37]. Once inside the cytoplasm, scopoletin would be glycosylated in scopolin and then stored in the vacuole with a pH of around 5.5. The chemical shifts of scopolin would this time shift downfield to a value close to that of scopoletin in the medium. This would explain why the signal becomes larger over time: the signals corresponding to scopoletin inside medium and scopolin inside vacuole resonating at the same place (left of the peak) and the signal corresponding to scopoletin inside cytoplasm (right of the peak) would actually overlap because too close to be separated in in vivo NMR spectra.

Another cross-peak visible in the TOCSY spectrum ([Fig. 3]) at 5.22/3.70 ppm (see upper enlargement) appears in parallel of the H3 – H4 cross-peak. It potentially corresponds to the correlation between H1′ and H2′ of the glucosyl moiety of scopolin and might reflect its biosynthesis from scopoletin and UDP-glucose, the latter being probably derived from sucrose, whose consumption can be followed via the two cross-peaks at 5.42/3.58 and at 5.42/3.79 ppm (see upper enlargement). However, the values of the chemical shifts of the glucosyl moiety of scopolin are not specific, and the H1′ and H2′ cross-peak might also correspond to many other glycosylated molecules. In order to follow specifically the time course of glycosylation of scopoletin into scopolin, it is necessary to visualize a correlation between one specific proton of the glucosyl moiety and one specific proton of the coumarin moiety. This could be achieved using a 2D NOESY analysis, which gives correlations between protons close in space but not necessarily presenting scalar coupling. [Fig. 5] shows the expansion of a NOESY spectrum obtained 30 h after the addition of scopoletin. In addition to the cross-peak at 7.08/3.86 ppm corresponding to the H5 and methoxy protons of scopolin and scopoletin, another cross-peak appears at 7.08/5.22 ppm. This latter cross-peak reflects specifically the biosynthesis time course of scopolin since it stems from the dipolar interaction between the H5 aglycone proton and the H1′ glycosyl proton of scopolin, thus confirming that the TOCSY cross-peak 5.22/3.70 ppm is due to scopolin and can be used to follow specifically the biosynthesis of this molecule.

[Fig. 6] shows the relative time courses, obtained from integrating some upper cross-peaks in the in vivo TOCSY spectra, for the sum of scopoletin and scopolin (H3 – H4 cross-peak), of scopolin alone (H1′-H2′ cross-peak), and of sucrose (glucosyl H1 – H2 cross-peak). It should be noted that the absolute height of the three curves cannot be compared as the TOCSY transfer efficiency is different. The scopol(et)in level increases linearly during the first 18 h and then reaches a near-plateau that decreases very slowly over time. During the accumulation phase, the scopolin level evolves in parallel with a lag time of about 4 h. Its decrease starts later, after 30 h, but is more pronounced. It is noticeable that the scopolin level starts decreasing when all the sucrose is consumed. This would suggest that scopolin is deglycosylated when sugar levels are low in the plant cell, probably in order to sustain energy metabolism.

These results indicate that even biotransformation can be followed by ¹H in vivo NMR, which implies no labeling. The in vivo monitoring of a glycosyl reaction could already be illustrated by a few examples [38], [39]. In the present study, it proved necessary to use 2D methods to provide the necessary spectral resolution and unambiguous identification of peaks. The drawback is that absolute peak levels cannot be compared, but relative evolution for a given peak is expected to be reliable. Absolute comparison could be possible by calibrating the curves with analysis of extracted samples. In general, compared to more conventional methods, in vivo metabolic NMR enables direct characterization of metabolic state of living cells or tissue of plant organs in a noninvasive way. It makes it possible to follow metabolic changes of plant samples in response to environment modifications. In addition, it is possible to follow stable isotopes as tracers and to obtain metabolic flux data. However, in vivo metabolic NMR has a low sensitivity, and due to the geometric constraints of the probe-head, it is often necessary to excise tissues, and there is a lack of lightening, which hampers the study of photodependent reactions.

Material and Methods

Cell suspensions of D. myoporoides

D. myoporoides cell suspensions were initiated from stem fragments of sterilized plants grown in a greenhouse. They were subcultured every 2 wk in 100 mL of Linsmaier and Skoog medium at pH 5.8, with addition of 2,4-D (0.2 mg/L), BAP (6-Benzyl-Amino-Purine) (0.1 mg/L), and sucrose (30 g/L) in 250-mL shake flasks. The cells were cultivated at 22 °C, on a rotary shaker at 100 rpm, under 2000 lux illumination with a 16-h photoperiod per day.

In vivo NMR device

A perfusion system, as shown in [Fig. 7], was used for this study. Three grams of 5-d-old wet D. myoporoides cells were placed in an 8-mm NMR tube. Perfusion was performed with 50 mL of oxygenated sugar-free culture medium driven by a peristaltic pump at a flow rate of 10 mL/min [34], [40]. At time 0 (after 4 h of perfusion), 15 mg of scopoletin in MeOH was added to obtain a final concentration of 1.6 mM in the culture medium. The total duration of the experiments was 2 d.

NMR analysis

NMR spectra were acquired on a Bruker Avance 300 WB equipped with an 8-mm BBO (Broad Band Observe) probe-head with z-gradients. The experiments were run without field lock, as the magnet was sufficiently stable. The temperature was kept constant at 298 K. The different spectra were recorded alternately.

Standard conditions for 1D ¹H spectra were a 90° pulse and a relaxation delay of 1.5 s, with water presaturation, for a total acquisition duration of 1 min 44 s. Conditions for 2D ¹H-¹H TOCSY spectra were a relaxation delay of 960 ms with water presaturation, a mixing time of 50 ms (MLEV-type spinlock), a F1 resolution of 23.5 Hz, for a total experiment duration of 58 min 29 s.

Conditions for 2D ¹H-¹H NOESY spectra recording were a relaxation delay of 800 ms with water presaturation, a mixing time of 300 ms, a F1 resolution of 23.5 Hz, for a total experiment duration of 59 min 30 s.

Spectra were referenced to the sucrose glucosyl H1 peak at 5.42 ppm.

Chemicals

Scopoletin (purity ≥ 99%) was purchased from Sigma. Scopolin was obtained by chromatographic purification after biotransformation of scopoletin to scopolin by D. myoporoides cell suspensions, according to the method described in [41].

Conflict of Interest

The authors declare no conflict of interest.

^* Dedicated to Professor Dr. Robert Verpoorte in recognition of his outstanding contribution to natural products research.

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Correspondence

• References

• 1 Osmani SA, Bak S, Møller BL. Substrate specificity of plant UDP-dependent glycosyltransferases predicted from crystal structures and homology modeling. Phytochemistry 2009; 70: 325-347
  CrossrefPubMedSearch in Google Scholar
• 2 Lao J, Oikawa A, Bromley JR, McInerney P, Suttangkakul A, Smith-Moritz AM, Plahar H, Chiu TY, Gonzalez-Fernandez-Ninno SM, Erbert B, Yang F, Christiansen KM, Hansen SF, Stonebloom S, Adams PD, Ronald PC, Hillson NJ, Hadi MZ, Vega-Sanchez ME, Loque D, Dcheller HV, Heazlewood JL. The plant glycosyltransferase clone collection for functional genomics. Plant J 2014; 79: 517-529
  CrossrefPubMedSearch in Google Scholar
• 3 Gachon CMM, Langlois-Meurinne M, Saindrenan P. Plant secondary metabolism glycosyltransferases: the emerging functional analysis. Trends Plant Sci 2005; 10: 542-549
  CrossrefPubMedSearch in Google Scholar
• 4 De Bruyn F, Maertens J, Beauprez J, Soetaert W, De Mey M. Biotechnological advances in UDP-sugar based glycosylation of small molecules. Biotechnol Adv 2015; 33: 288-302
  CrossrefPubMedSearch in Google Scholar
• 5 Sucha L, Tomsik P. The steroidal glycoalkaloids from Solanaceae: toxic effect, antitumour activity and mechanism of action. Planta Med 2016; 82: 379-387
  Thieme ConnectPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 14 Fourcroy P, Siso-Terraza P, Sudre D, Saviron M, Reyt G, Gaymard F, Abadia A, Abadia J, Alvarez-Fernandez A, Briat JF. Involvement of the ABCG37 transporter in secretion of scopoletin and derivatives by Arabidopsis roots in response to iron deficiency. New Phytol 2014; 201: 155-167
  CrossrefPubMedSearch in Google Scholar
• 15 Siwinska J, Kadzinski L, Banasiuk R, Gwizdek-Wisniewska A, Olry A, Banecki B, Lojkowska E, Ihnatowixz A. Identification of QTLs affecting scopolin and scopoletin biosynthesis in Arabidopsis thaliana . BMC Plant Biol 2014; 14: 280
  CrossrefPubMedSearch in Google Scholar
• 16 Ziegler J, Schmidt S, Chutia R, Mueller J, Boettcher C, Strehmel N, Scheel D, Abel S. Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation. J Exp Bot 2016; 67: 1421-1432
  CrossrefPubMedSearch in Google Scholar
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