Galanthamine, an anti-cholinesterase drug, effects plant growth and development in Artemisia tridentata Nutt. via modulation of auxin and neurotransmitter signaling

Abstract

Galanthamine is a naturally occurring acetylcholinesterase (AchE) inhibitor that has been well established as a drug for treatment of mild to moderate Alzheimer disease, but the role of the compound in plant metabolism is not known. The current study was designed to investigate whether galanthamine could redirect morphogenesis of Artemisia tridentata Nutt. cultures by altering concentration of endogenous neurosignaling molecules acetylcholine (Ach), auxin (IAA), melatonin (Mel), and serotonin (5HT). Exposure of axenic A. tridentata cultures to 10 µM galanthamine decreased the concentration of endogenous Ach, IAA, MEL, and AchE, and altered plant growth in a manner reminiscent of 2–4D toxicity. Galanthamine itself demonstrated IAA activity in an oat coleoptile elongation bioassay, 20 µM galanthamine showed no significant difference compared with 5 μM IAA or 5 μM 1-Naphthaleneacetic acid (NAA). Metabolomic analysis detected between 20,921 to 27,891 compounds in A. tridentata plantlets and showed greater commonality between control and 5 µM treatments. Furthermore, metabolomic analysis putatively identified coumarins scopoletin/isoscopoletin, and scopolin in A. tridentata leaf extracts and these metabolites linearly increased in response to galanthamine treatments. Overall, these data indicate that galanthamine is an allelopathic phytochemical and support the hypothesis that neurologically active compounds in plants help ensure plant survival and adaptation to environmental challenges.

Introduction

The classic hypothesis of the regulation of plant growth and regeneration attributes morphological development to changes in the relative ratio of auxins (IAA) to cytokinins.¹ Interpretations and applications of this approach are the foundation of modern plant tissue culture and biotechnology. However, not all plants respond in the same way as model systems such as tobacco, and other plant growth regulators have been discovered that mediate, accelerate, or enhance fundamental plant regeneration mechanisms, especially in medicinal plants.² In particular, human neurosignaling compounds have been shown to influence plant metabolism, growth, and development.^3-5 In plant tissue culture, the capacity for neurologically active phytochemicals to enhance cell survival and regeneration warrants further study.

Artemisia tridentata Nutt and its subspecies are collectively known as Big Sagebrush and are found in arid regions of North America from steppe to subalpine zones, dry shrub lands, foothills, rocky outcrops, scablands, and valleys.^6,7 Traditionally, species of Big Sagebrush have been used as a ceremonial medicine to treat headaches or protect individuals from metaphysical forces.⁸ A total of 220 phytochemicals have been described in A. tridentata and related species in the Tridentatae.⁹ Recently, the neurologically active compounds melatonin (MEL), serotonin (5HT), and acetylcholine (Ach) were identified and quantified in axenic cultures of A. tridentata.¹⁰ MEL and 5HT have previously been identified and quantified in axenic culture of several plant species.^11-14 Tryptophan was identified as a precursor for biosynthesis of MEL and 5HT in Hypericum perforatum¹¹ and key enzymes and genes in this pathway have been described in rice.¹⁵ Current research indicates that MEL and 5HT may be important in plant metabolic systems such as photosynthesis¹⁶ and physiological responses to environmental stresses such as tolerance to cold stress.^17-19 Ach was first described in plants in the early 1970s using bioassays and detected in plants by gas chromatography in 1974.²⁰ Ach occurs in all parts of plants, but higher concentrations have been quantified in roots and dark-adapted tissues than in shoots or light-exposed cells.^20-23 The hypothesized roles of Ach in plants include almost all aspects of growth and development,²³ including phytochrome-linked signaling,²⁴ phytochrome-modulated calcium signaling,²⁵ induction of primary and secondary rooting,²⁶ regulation of phloem transport,²⁷ and regulation of pollen tube development.²⁸

Previous work has shown that many synthetic and natural drugs that affect human neurotransmitter metabolism can also mediate plant growth and development.^4,12,13 In order to investigate the potential role of the cholinergic system in plant growth and development, we hypothesized that the acetylcholinesterase (AchE) inhibiting natural product galanthamine could redirect morphogenesis in A. tridentata cultures by altering the concentration of endogenous neurosignaling molecules such as Ach, MEL, 5HT, and IAA. To investigate this hypothesis, we designed the following specific objectives: 1) quantify neurosignaling molecules in axenic cultures of A. tridentata, 2) determine whether inhibition of plant cholinergic signaling affects MEL, 5HT, and/or IAA in A. tridentata, and 3) develop and analyze a metabolomic data set as a hypothesis generating tool for abiotic stresses.

Results

In vitro regeneration

A. tridentata shoot proliferation was sustained on basal MSO medium, and altered morphologies were observed in response to galanthamine (Fig. 1A and 1B). Treatment with 5 μM galanthamine appeared to increase leaf length and reduce turgor pressure. Cultures treated with 10 µM galanthamine appeared chlorotic, exhibited leaf curling, and were vitrified compared with the control or 5 µM treatments (Fig. 1B).

Figure 1. (A) Effect of galanthamine on the growth and development of Artemisia tridentata cultures. (a) 0 µM galanthamine. (b) 5 µM galanthamine. (c) 10 µM galanthamine. (B) Effect of 10 µM galanthamine on growth and development of Artemisia tridentata cultures.

Ach and acetycholinesterase activity

Ach concentration in A. tridentata cultures was significantly lower in plants treated with 10 µM galanthamine (45.9 ± 3.01 µg/g) compared with control plants (92.8 ± 12.6 µg/g) or 5 µM galanthamine treated plants (98.4 ± 3.01 µg/ g) (Fig. 2A). Exposure of plant tissues to 10 µM galanthamine also significantly decreased the activity of AchE in the tissues (454.1 ± 19.8 mU/g) as compared with the control (1004.6 ± 178.4 mU/ g) and 5 µM galanthamine treatments (1129.1 ± 207.3 mU/ g) (Fig. 2B).

Figure 2. Effect of galanthamine on (A) Ach and (B)AchE concentration.

Effects of galanthamine on IAA and indoleamines

Regression analysis demonstrated a significant negative linear relationship (R² = 0.991) between the concentration of galanthamine and their endogenous levels of MEL. Significant differences were found between the control (9.6 ± 3.1 ng/g) and 10 µM treatments (2.3 ± 0.9 ng/g) (Fig. 3A). Interestingly, 5HT levels were significantly lower in plants exposed to 5 µM galanthamine (10.7 ± 3.6 ng/g) compared with controls (37.1 ± 16.5 ng/g) or the 10 µM treatment (35.4 1 ± 15.3 ng/g) (Fig. 3B). Endogenous IAA levels also demonstrated a negative linear relationship with galanthamine treatment (R² = 0.9989), but the overall differences between treatments were not significant (Fig. 3C).

Figure 3. Effect of galanthamine on indoleamine and IAA in Artemisia tridentata cultures. (A) MEL. (B) 5HT. (C) IAA.

Analysis of oxidative stress in galanthamine treated cultures

Quantification of the antioxidant potential of control and galanthamine-treated tissues using a DPPH microplate assay indicated that significantly less tissue was required to quench DPPH by 50% in 5 µM (473 ± 42 µg) and 10 µM (529 ± 87 µg) treatments as compared with the control (930 ± 83 µg) (Fig. 4). This demonstrates that plates treated with galanthamine had a higher antioxidant potential than control plants.

Figure 4. Effect of galanthamine on the antioxidant capacity of Artemisia tridentata tissues grown in vitro.

Activity of galanthamine in an IAA bioassay

Inclusion of galanthamine in the solution significantly increased the elongation of oat coleoptiles in a manner similar to the classic IAA response (Fig. 5). Exposure of coleoptiles to 20 μM galanthamine significantly increased elongation as compared with controls and was not significantly different from 5 μM IAA or 5 μM 1-Naphthaleneacetic acid (NAA).

Figure 5. Galanthamine showingIAA-like activity in an oat coleoptile bioassay.

Untargeted phytochemical analysis on galanthamine treated cultures

The metabolomes of A. tridentata shoot cultures contained more than 20,000 distinct compounds (Table 1) with about 27,000 detected in controls and 5 µM galanthamine treatments. Fewer compounds were detected in the 10 µM galanthamine treated tissues than the control or 5 μM galanthamine treatments. Scores plots for PC-1(23.12%) and PC-2 (19.28%) showed significant clustering for 10 µM treated cultures in contrast to the significant overlap observed between the control and 5 µM cultures (Fig. 6). Of the 20921–27891 compounds found, 161 had average ion intensities greater than 5.0 (> 0.09% of average total ion count). Linear or exponential responses to galanthamine treatments were observed for 32 (decreasing) and 50 (increasing) compounds (Table 2). Putative identifications were performed for all prominent compounds (data not shown). The most significant findings were the putative identification of coumarins (scopoletin/isoscopoletin and scopolin) and their precursor phenylalanine as compounds significantly impacted by the exposure to galanthamine (Table 2). The average ion intensity of phenylalanine (m/z = 165.0790) significantly decreased (R² = 0.9986), while the ion intensities of scopoletin/isoscopoletin (m/z = 192.0423, R² = 0.8044) and its glucocylated derivative scopolin (m/z = 354.0951, R² = 0.9561) increased in response to galanthamine treatments (Fig. 7).

Table 1. Summary of Metabolomic Data of Galanthamine Treated Cultures.

	Control	5 µM Galanthamine	10 µM Galanthamine
Total Number of compounds	27891	27851	20921
Average Number of Compounds	16663 +/− 241	17083 +/− 491	13786 +/− 192
Compounds Common to All	6219	6799	4798
Compounds Unique Between Treatments
	Control	5 µM Galanthamine	10 µM Galanthamine
10 µM Galanthamine	2291	2213	0
5 µM Galanthamine	9137	0	2213

Figure 6. Principle Component Analysis identifying the characteristic responses of axenic cultures of Artemisia tridentata to galanthamine treatment.

Table 2. Most Prominent Ions (Average Ion Counts > 5.0 (0.09%)) Showing Linear or Exponential Trends.

Decreasing with Treatment
ID	Ret. Time	Mass	Line of Best fit	Equation	R2
1	6.9558	340.0927	Linear	y = -0.2425x + 29.744	0.9929
2	6.0175	241.0592	Linear	y = -0.2425x + 29.744	0.9929
3	13.5308	334.1383	Linear	y = -2.1247x + 28.83	0.8788
4	1.7363	213.0418	Linear	y = -1.0884x + 21.545	0.9578
5	8.9819	434.1102	Exponential	y = 22.657e^-0.087x	0.9545
6	6.7	233.113	Exponential	y = 16.501e^-0.016x	0.9827
7	1.8035	258.0591	Linear	y = -2.3942x + 26.101	0.9964
8	13.5301	233.1084	Linear	y = -1.4829x + 21.035	0.8013
9	6.0152	276.0961	Linear	y = -0.4833x + 14.438	0.9926
10	6.2874	227.0927	Linear	y = -0.7726x + 15.239	0.9506
11	6.0038	517.1004	Linear	y = -1.4169x + 18.422	0.9510
12	15.1925	505.1853	Linear	y = -1.4143x + 15.892	0.8489
13	1.6532	349.1665	Exponential	y = 16.153e^-0.169x	0.9935
14	7.4304	480.1565	Linear	y = -0.6014x + 11.605	0.9843
15	9.9656	434.1394	Exponential	y = 9.5567e^-0.04x	0.9144
16	15.2634	375.0408	Linear	y = -0.6859x + 11.272	0.9619
17	8.5777	399.0983	Exponential	y = 9.3338e^-0.046x	0.9243
18	6.6231	352.0954	Linear	y = -0.2273x + 8.1602	0.9322
19	9.8383	345.0929	Linear	y = -0.3421x + 8.5447	0.9997
20	12.416	284.1271	Exponential	y = 18.081e^-0.391x	0.9544
21	5.658	406.0925	Linear	y = -0.8878x + 10.342	0.9593
22	6.0164	205.0516	Linear	y = -0.2074x + 6.8009	0.9392
23	2.0929	284.0493	Linear	y = -0.5524x + 8.4621	0.9663
24	14.6013	284.1245	Linear	y = -0.6884x + 9.1244	0.9908
25 A	3.9644	166.0617	Linear	y = -0.14x + 6.3751	0.9986
26	8.6167	210.11	Exponential	y = 7.8604e^-0.079x	0.9960
27	8.9827	399.0978	Exponential	y = 8.0114e^-0.086x	0.9980
28	1.7025	387.1295	Linear	y = -0.7391x + 9.0713	0.9968
29	8.4876	169.1004	Linear	y = -0.6547x + 8.467	0.7922
30	22.367	235.1366	Linear	y = -0.7584x + 8.9401	0.9744
31	10.7236	582.172	Linear	y = -0.7747x + 8.91	0.9526
32	9.8427	327.0888	Linear	y = -0.2612x + 6.339	0.9882
Increasing with Treatment
ID	Ret. Time	Mass	Line of Best fit	Equation	R2
21	9.5537	199.0965	Linear	y = 1.1095x + 34.389	0.9410
22	1.8738	380.991	Linear	y = 1.3206x + 33.245	0.9778
23	13.9895	215.116	Linear	y = 1.1765x + 31.268	0.9722
24	6.3407	245.0937	Exponential	y = 28.216e^0.0427x	0.7988
25	5.61	355.004	Linear	y = 1.8953x + 14.458	0.9560
26	5.1247	205.0568	Exponential	y = 5.5215e^0.1735x	0.8681
27	8.4323	354.1065	Linear	y = 0.2937x + 17.805	0.8137
28	8.5697	516.9717	Linear	y = 3.1768x - 0.6695	0.9845
29 B	5.3907	193.0583	Exponential	y = 8.7431e^0.0777x	0.8284
30	7.9198	399.0968	Exponential	y = 8.8366e^0.078x	0.9997
31	5.6303	355.0657	Linear	y = 0.7487x + 7.7082	0.9981
32	5.3821	390.1043	Exponential	y = 7.8235e^0.0536x	0.8414
33	5.1447	341.0321	Exponential	y = 5.4765e^0.1026x	0.8517
34	9.8245	523.021	Exponential	y = 2.0228e^0.2333x	1
35	1.742	290.7999	Linear	y = 0.9645x + 4.58	0.9966
36 C	5.5567	355.0838	Linear	y = 0.5845x + 5.6911	0.9561
37	11.4011	453.0584	Exponential	y = -0.6014x + 11.605	0.9843
38	7.8306	534.1533	Linear	y = 0.4806x + 5.0447	0.7993
39	5.1249	188.0433	Exponential	y = 1.9949e^0.1862x	0.8455
40	1.8441	218.9918	Exponential	y = 3.5563e^0.1186x	0.9998
41	1.5434	184.9998	Linear	y = 0.3741x + 5.2801	0.9420
42	5.131	246.0826	Exponential	y = 2.4741e^0.1565x	0.7967
43	8.0024	194.0502	Exponential	y = 4.3066e^0.0751x	0.7676
44	22.1758	593.2024	Linear	y = 1.3187x - 0.1007	0.9993
45	12.5212	469.1415	Exponential	y = 1.108e^0.2394x	0.9468
46	11.8184	418.1451	Exponential	y = 4.0479e^0.06x	0.8826
47	12.2741	416.1586	Exponential	y = 0.3947e^0.3309x	0.9005
48	6.3831	422.0663	Exponential	y = 2.3529e^0.1352x	0.9879
49	1.5636	122.5363	Linear	y = 0.1846x + 4.1701	0.9528
50	9.9414	659.1121	Exponential	y = 1.3175e^0.1987x	0.9541

^*Putative Identifications: A = Phenylalanine, B = Scopoletin/Isoscopoletin, C = Scopolin*

Figure 7. Putative identification of prominent ions with linear responses to galanthamine treatment.

Discussion

Galanthamine is a natural inhibitor of AchE, first isolated from snowdrop (Galanthus spp) and commonly used in the treatment of Alzheimer disease.²⁹ In humans, galanthamine increases Ach levels in the brain by reversibly inhibiting AchE, while increasing sensitivity of neuronal nicotinic receptors to surrounding Ach.³⁰ The initial observation of Ach in Artemisia tissues¹⁰ prompted studies to investigate the role of Ach in plant growth and development. The most significant findings of the current investigations were the degree and diversity of responses of A. tridentata tissues to galanthamine exposure. There was an overall inhibition of the cholinergic system with decreases in both Ach and AchE in response to galanthamine. In addition, there was a decrease in IAA and MEL in the galanthamine treated A. tridentata but the effects on plant 5HT were less clear. Interestingly, galanthamine induced a clear IAA-like growth response in the classic oat coleoptile IAA bioassay and the galanthamine treated A. tridentata plants appeared elongated, chlorotic, and vitrified. The metabolomic analysis identified the increase of polyphenols as the most significant phytochemical response to galanthamine. Together these data indicate the induction of a cascade of responses that include growth regulation, stress, and the induction of defense mechanisms by the natural product.

Cholinergic system effects

It has been proposed that Ach regulates basic cellular functions that are required for survival in vertebrates, invertebrates, plants, fungi, and mosses.^31-33 Ach has been reported in many plant species but specific physiological roles remain under debate.²³ Our data indicated that an overall decrease in cholinergic metabolism in A. tridentata manifested mainly as stress in the tissues. Bamel et al. (2007) found that the application of exogenous Ach or inhibitors of its breakdown induced rhizogenesis in tomato cultures (Lycopersicon esculentum Miller), which led them to propose that Ach may interact with other plant regulators such as IAA or MEL.²⁶ Our data support this observation by demonstrating an interaction between galanthamine and IAA. The observation that oat coleoptiles elongated in response to galanthamine could indicate that the mechanism of action may involve cross talk between IAA and Ach pathways.

IAAs and indoleamines

Regulation of shoot and root development by 5HT and MEL was previously investigated with a variety of pharmaceuticals used to regulate neuronal 5HT metabolism in humans^3,4,12 and served as a model for the current study. Several researchers have proposed roles for MEL and 5HT in mediation of stress including protection of plant organelles and structures from oxidative damage caused by free radicals^16,34-36 and the elimination of extraneous metabolic products through detoxification.³⁷ The current data are the first suggestion of synergy between cholinergic metabolism and neuroindoleamine signaling in plants. In human systems, the interaction between cholinergic and serotonergic systems during cognitive processes has been well documented,³⁸ and 5HT receptor subtypes are under investigation as potential drug targets for diseases such as Alzheimer.³⁹

Metabolomics as a hypothesis generating tool

Metabolomics is the qualitative and quantitative analysis of all small metabolites in a biological sample.^40,41 In recent years, the application of metabolomics has provided an interesting avenue for understanding how plants respond to specific environmental stressors.^42,43 Understanding how the plant metabolome responds to neurologically active phytochemicals has not been investigated, but environmental cues such as light, temperature, drought, or salt stresses have been examined⁴⁴ with broader applications toward crop selection.^45,46 There are 3 distinctly different research approaches that are taken in metabolomics research. Some researchers are using a strictly targeted approach with quantification of known compounds using authenticated standards and spectral libraries sometimes associated with genomic or proteomic data.⁴⁷ Other researchers are using the genomic or proteomic data as chemical leads in a pseudo-untargeted approach that quantifies known compounds from other species but that have not previously been described in the species of interest. These data may be mapped in association with the genomic data for compound and pathway discovery but are not truly untargeted analyses.⁴⁸ The third approach is completely untargeted identification and quantification of the entire metabolome using statistics modeling and mapping to eliminate false discoveries, explore relationships between unknowns, and to target specific characteristic unknowns for fingerprinting, future targeted phytochemical studies, pathway elucidation, chemical association with physiological responses, and other applications.⁴⁹

In the current work, we investigated changes in the whole plant metabolome in response to galanthamine treatment. Untargeted analysis identified phenylpropanoid metabolism as a primary response for further study. Coumarins are a class of secondary metabolites derived from the shikamate pathway via phenylalanine and contribute to plant defense through their anti-fungal and anti-microbial properties.⁵⁰ Previous research identified coumarins, including scopoletin, isoscopoletin, esculin, esculetin, methylesculin, and umbelliferone, in leaf extracts of A. tridentata.^51,52 Our exploratory analysis found increasing amounts of scopoletin and its monoglucoside scopolin in response to galanthamine exposure. Increased levels of scopoletin and scopolin have been shown to occur in response to stresses such as microbial attack, water stress, and wounding.⁵⁰ Furthermore, manipulation of cultures with growth regulators such as 2–4D, kinetin, salicylic acid, and methyl jasmonate was previously found to affect levels of these compounds,⁵⁰ most notably in tobacco cultures.^53-55 Although more enquiry is needed, our untargeted exploratory analysis has allowed us to formulate the hypothesis that cultures treated with galanthamine increase production of phenolic compounds.

Conclusions and implications

Galanthamine is a very interesting natural drug that is found in a wide range of species including snowdrops, Narcissis, and other common bulbs. The utility of galanthamine for modulation of human neurochemistry is well established but less is known about the role of galanthamine in plant metabolism. Our data indicate a role for galanthamine as an allelopathic phytochemical released by one species into the environment and inducing a wide spectrum of responses in the phytochemistry of other plants. In order to survive abiotic and biotic environmental stresses, plants use many phytochemicals, including neurotransmitters or neurologically active signaling molecules, and our data indicate this potential role for IAA, indoleamines, and polyphenols. Previous researchers have hypothesized that these neuro-mimicking plant responses indicate plant communication, plant neurosignaling, or plant avoidant behaviors necessary for survival.^56,57 The systems approach to generating novel phytochemical hypotheses through metabonomic analysis provides new avenues for future studies of the responses of plant tissues to environmental stimuli, allelochemicals, and natural products.

Materials and Methods

Establishment of germplasm lines

A collection of axenic stock plants of A. tridentata plants were established from individual seeds in vitro with previously established protocols¹⁰ and maintained on a sterile medium hereafter referred to as “MSO,” containing 4.4g/L Murashige and Skoog salts,⁵⁸ B5 vitamins,⁵⁹ and 30 g/l sucrose at pH 5.7. 3g/L phytagel was added to all media prior to autoclaving for 20 min at 121 °C at 1.4 x 10⁴ kg/m/s². Germplasm lines were established by regeneration of shoot cultures from individual seedlings as described previously,¹⁰ maintained at standard temperature (28 °C) with a 16 h photoperiod (35–50 μmol/m²/s) and sub-cultured on MSO medium into GA7 boxes (Magenta Corp) at 2 wk intervals.

Galanthamine treatments

Shoot tissue from the A. tridentata germplasm line DCO-1–09 were sub-cultured onto MSO medium in petri dishes (n = 12) (see ref. 30). For each treatment (n = 4), a solution of galanthamine hydrobromide (1.0 mL of 0, 5, or 10 µM; Chromadex, Vancouver) was filter sterilized (Millipore PVDF, 0.22 μm, 25 mm sterile syringe filters; Fisher Scientific, USA) and layered onto the sterile media twice weekly for a period of 1 month to ensure a continual dose. At the end of the experiment, tissues were excised, flash frozen and stored at –80 ^◦C until further testing.

Preparation of extracts

A method was developed for analysis of neurotransmitters, IAA, and antioxidant potential in the same extract to ensure valid data comparison. In brief, an explant of A. tridentata leaf tissue was extracted in MeOH (0.5 g/mL) in complete darkness and an aliquot of 100 µL of each sample was dried under nitrogen gas and re-suspended in 18 mΩ water (Millipore). Remaining MeOH extract was stored at –80 ^◦C for further analysis.

Quantification of ach and acetylcholinesterase

The cholinergenic system was quantified with an Amplex© Red Ach / Acetylcholinesterase Assay Kit (Molecular Probes Inc, Eugene, Oregon).^60,61 Ach was quantified in tissues using a modification of the spectrophotometric method originally described in 1961.⁶² In brief, 100µL of Ach standard (n = 3) (0, 1, 5, 10, 20, 40, 60, 80, and 100µM) or AchE standard (n = 3) (0, 1, 2.5, 5, 10, 15, 25, 50, and 100 mu/mL), positive/negative control (n = 4), and A. tridentata tissue sample (0.5 g/mL) were added to a 96 well plate. All samples were prepared in 1X buffer. 100 µL of working solution (200 µM Amplex red reagent, 1 U/mL Horseradish peroxidase, 0.1 U/mL choline oxidase, 0.5 U/mL AchE, or 50 µM Ach, Buffer) was added to each well, lightly shaken and placed into a Synergy multi-detection microplate reader (BioTek Instruments Inc, USA), with excitation and emission wavelengths set to 530–560 nm and 590 nm, respectively. Fluorescence was measured every 5 min until the reaction was complete (1 h). A standard curve for Ach and AchE was generated by subtracting standards by the negative control (0 µM Ach or AchE). Ach and AchE were then quantified for each sample by subtracting the observed fluorescence values for each sample by the negative control and sample blank and comparing to standard curve data.

Analysis of MEL, 5HT, and IAA

IAA and indoleamines were separated from a 1 µL aliquot of the extract on a reverse phase column (150 x 2.1 mm, 1.7 μm C18 BEH, Waters) using a Waters I-Class UPLC. A gradient of 0.1% formic acid (Eluent A) and acetonitrile (Eluent B) [(A%:B%): 0.0–0.5 min, 99:1; 0.5–2.5 min, 70:30; 2.5–2.6 min, 5:95; 2.6–3.0 min, 5:95; 3–3.5 min, 5:95; 3.5–3.6 min, 99:1; 3.6–5.0 min, 99:1] separated MEL, 5HT, and IAA with a flow rate of 0.5 ml/min. Analytes were detected and quantified with a tandem mass spectrometer (Xevo TQ-S triple quadrupole mass spectrometer, Waters, Mississauga) using detection parameters optimized for signal intensity and specificity (Table 3). The capillary voltage was 3500, desolvation gas rate was 800 L/hr, cone gas rate was 150 L/hr, desolvation temperature was 400 °C, and the source temperature was 150 °C for all analyses. Authentic standards were injected over a wide range to establish the limits of detection (LOD) and limits of quantification (LOQ). The linear ranges were 1–100 ng/mL for MEL and 1–500 ng/mL for 5HT and IAA. In preliminary experiments it was determined that a gradient of IAA and indoleamine was present across the plant tissues. Therefore, to determine the response to galanthamine, plantlets were subsampled by sectioning into 3–5 pieces (259 ± 10 mg) prior to analysis and each subsample was analyzed in triplicate.

Table 3. Optimized detection of Indoleamines and IAA by Tandem Mass Spectrometry (Waters Xevo TQ-S).

	Cone voltage (V)	Collision voltage (V)	MRM Transition
IAA	30	25	176 > 103
IAA	30	13	176 > 130
5HT	45	27	177 > 115
5HT	45	10	177 > 160
MEL	30	23	233 > 159
MEL	30	15	233 > 174

IAA and indoleamine data analysis

Indoleamines and IAA were quantified by comparison to the linear range of standard curves established with authenticated standards (Sigma, USA). Data for each plantlet were determined by a proportioned approach. In brief, the fresh weight of each excised section was divided by the total weight of the plantlet it originated from in order to scale the proportionate MEL, 5HT, and IAA concentrations in the entire tissues.

Antioxidant potential

Antioxidant potential was determined from the same extracts with a DPPH bioassay as described previously.⁶³ In brief, the extracts were diluted (n = 6), replicated (n = 24) and randomly assigned to a 96 well microplate alongside extract blanks (n = 24), assay blanks (n = 3), negative control (n = 3), and a positive control of Trolox ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; Sigma, purity ≥ 97%) dilutions (1, 8, 16, 24, 32, 40, and 50 µM) in triplicate. An aliquot of each extract or Trolox standard (100 µL) reacted with 100 µL of 0.1 mM DPPH for 15 min at 25 °C. Absorbance was recorded every 60s at 520 nm using a Synergy multi-detection microplate reader. Radical scavenging activity was calculated at t = 15 min and expressed as required to reduce free radicals by 50%.

IAA bioassay

IAA activity was determined using an oat (Avena sativa) coleoptile elongation bioassay as previously described.⁶⁴ In brief, coleoptile tips (about 0.3 mm) were removed and 10 mm sections were incubated in 10 ml of sterile distilled water supplemented with various concentrations of galanthamine (0, 0.05, 0.1, 1, or 10 μM), NAA (5 μM), or IAA (5 μM) in 15 mm petri plates. After 24 h, the coleoptiles were measured again to determine the degree of elongation. Each plate contained 10 sections and each treatment was replicated thrice for a total of 30 sections per treatment. The plates were arranged in a completely randomized design and the experiment was conducted twice. Significance between treatments was determined using the Tukey means separation test (α=0.05).

Metabolomic analysis

A. tridentata shoot cultures treated with 0, 5, and 10 µM of galanthamine were sampled in triplicate and subjected to untargeted phytochemical analysis using previously published protocols.^49,66 In brief, shoot tissues were extracted in 70% ethanol (0.05 g/mL), homogenized, and filtered by centrifugation (UltraFree MC, Millipore). Extracts were then separated on a Waters BEH Acquity C₁₈ (2.1 X 150 mm, 1.7 µm) column with the following gradient: 0.1% aqueous formic acid:acetonitrile (0.0–25 min, 95:5–5:95 v/v, 25.01–30.0 min, 95:5 v/v). The flow rate was set to 0.25 mL/min for 30 min at 30 °C (Waters Acquity UPLC). A steady flow of leucine enkephalin (Waters 1525 HPLC binary solvent manager, 2 ng/mL) was used as the internal standard for calibration of the Micromass LCT Premier series ToF-MS (Waters Inc). Time of Flight mass spectrometry used previously published optimized conditions including: electrospray ionization and positive and negative ion detection in W mode, mass range of 100–1000 amu and a scan time of 0.1 s. Data were collected with MassLynx V4.1 and exported via MarkerLynx. Data were processed in Excel (Microsoft) to align retention times and remove multiply charged ions as described previously.^49,65

Analysis of metabolomic data

Average ion intensity was calculated for each compound in order to select for metabolites that are most prominent throughout all treatments and replicates. Once ion averages were calculated, they were divided by the average total ion count in order to identify compounds that possess average ion intensities greater than 5.0 (0.09% of the average total ion intensity). Using Excel, average ion intensity for each prominent ion (dependent) was plotted against 0, 5, and 10 µM galanthamine treatments (independent), in order to determine whether a linear relationship exists. Compounds exhibiting exponential or linear behavior (R² > 0.75) in response to galanthamine treatments were then subjected to putative identification using Metabosearch http://omics.georgetown.edu/MetaboSearch.html, Plant Metabolic Pathway Database http://www.plantcyc.org/, and phytochemical information gathered from a comprehensive review on North American Sagebrush.⁹ Putative identifications (± 0.02 daltons) were performed using a previously published protocol^63,66 (Figs. 8 and 9).

Glossary

Abbreviations:

Ach

Ach

AchE

Acetylcholinesterase

IAA

Auxin

MEL

Melatonin

5HT

Serotonin

Disclosure of Potential Conficts of Interest

No conflicts of interest have been disclosed.