Tyramine pathways in citrus plant defense: Glycoconjugates of tyramine and its N-methylated derivatives

Article abstract of DOI:10.1021/acs.jafc.6b04423

Glucosylated forms of tyramine and some of its N-methylated derivatives are here reported for the first time to occur in Citrus genus plants. The compounds tyramine-O-β-D-glucoside, N-methyltyramine-O-β-D-glucoside, and N,N-dimethyltyramine-O-β-D-glucoside were detected in juice and leaves of sweet orange, bitter orange, bergamot, citron, lemon, mandarin, and pomelo. The compounds were identified by mass spectrometric analysis, enzymatic synthesis, and comparison with extracts of Stapelia hirsuta L., a plant belonging to the Apocynaceae family in which N,N-dimethyltyramine-O-β-D-glucoside was identified by others. Interestingly, in Stapelia hirsuta we discovered also tyramine-O-β-D-glucoside, N-methyltyramine-O-β-D-glucoside, and the tyramine metabolite, N,N,N-trimethyltyramine-O-β-glucoside. However, the latter tyramine metabolite, never described before, was not detected in any of the Citrus plants included in this study. The presence of N-methylated tyramine derivatives and their glucosylated forms in Citrus plants, together with octopamine and synephrine, also deriving from tyramine, supports the hypothesis of specific biosynthetic pathways of adrenergic compounds aimed to defend against biotic stress.

Full text of DOI:10.1021/acs.jafc.6b04423

Article
pubs.acs.org/JAFC
Tyramine Pathways in Citrus Plant Defense: Glycoconjugates of
Tyramine and Its N‑Methylated Derivatives
Luigi Servillo,*,† Domenico Castaldo,‡,§ Alfonso Giovane,† Rosario Casale,† Nunzia D’Onofrio,†
Domenico Cautela,‡ and Maria Luisa Balestrieri†
†Department of Biochemistry, Biophysics and General Pathology, Universita
Crecchio 7, 80138, Naples, Italy
̀
degli Studi della Campania “Luigi Vanvitelli”, via L. De
‡Stazione Sperimentale per le Industrie delle Essenze e dei derivati dagli Agrumi, Azienda Speciale della Camera di Commercio di
Reggio Calabria, Via Generale Tommasini 2, 89127 Reggio Calabria, Italy
§Ministero dello Sviluppo Economico, Via Molise 2, Roma, Italy
ABSTRACT: Glucosylated forms of tyramine and some of its N-methylated derivatives are here reported for the first time to
occur in Citrus genus plants. The compounds tyramine-O-β-D-glucoside, N-methyltyramine-O-β-D-glucoside, and N,N-
dimethyltyramine-O-β-D-glucoside were detected in juice and leaves of sweet orange, bitter orange, bergamot, citron, lemon,
mandarin, and pomelo. The compounds were identified by mass spectrometric analysis, enzymatic synthesis, and comparison
with extracts of Stapelia hirsuta L., a plant belonging to the Apocynaceae family in which N,N-dimethyltyramine-O-β-D-glucoside
was identified by others. Interestingly, in Stapelia hirsuta we discovered also tyramine-O-β-D-glucoside, N-methyltyramine-O-β-D-
glucoside, and the tyramine metabolite, N,N,N-trimethyltyramine-O-β-glucoside. However, the latter tyramine metabolite, never
described before, was not detected in any of the Citrus plants included in this study. The presence of N-methylated tyramine
derivatives and their glucosylated forms in Citrus plants, together with octopamine and synephrine, also deriving from tyramine,
supports the hypothesis of specific biosynthetic pathways of adrenergic compounds aimed to defend against biotic stress.
KEYWORDS: Citrus plant, tyramine-glucoside, N-methyltyramine-glucoside, hordenine-glucoside, candicine-glucoside, Stapelia hirsuta,
biotic stress
catalyzed by the enzyme tyrosine decarboxylase (TYDC),
producing tyramine, from which octopamine (4-(2-amino-1-
hydroxy-ethyl)phenol) is derived by subsequent hydroxylation
by the enzyme tyramine-β-hydroxylase (TβH).3 Both tyramine
and octopamine, which exert adrenergic effects in mammals, are
able to function in plants as neurotransmitters and neuro-
modulators by acting on specific receptors of plant attackers
such as insects and herbivores. In this way, these compounds
modify behavior and metabolism of plant attackers through the
activation of G protein-coupled receptor-mediated signal
transduction pathways.5−8 Four octopamine receptors (Oamb,
Octβ1R, Octβ2R, Octβ3R) and three tyramine receptor (TyrR,
TyrRII, TyrRIII) modulate metabolic functions and behavior of
invertebrates, such as flying ability, learning capacity, memory,
and sleep/wake cycle, and also influence fertility.7,9−11 In some
plant genera, including Citrus genus,12−16 successive N-
methylation of tyramine by the same or different methyl-
transferase(s) produces N-methyltyramine and N,N-dimethyl-
tyramine (hordenine). In citrus plants, synephrine (N-
methyloctopamine), a powerful adrenergic amine, originates
from the hydroxylation of N-methyltyramine, whereas in
animals it is formed through the N-methylation of octop-
amine.14
INTRODUCTION
■
The enzymatic decarboxylation of the aromatic amino acids
phenylalanine, tyrosine, and tryptophan in plants represents a
physiological process which operates as an interface between
the primary and secondary metabolism aimed at production of
defensive substances in response to attacks by insects,
herbivores, and pathogens.1 In citrus plants, two pathways for
the biosynthesis of secondary metabolites, which likely play
defensive roles, have been recently highlighted.2,3 The first
pathway starts with tryptophan decarboxylation by the enzyme
trypthophan decarboxylase (TDC), producing the biogenic
amine tryptamine, which then, by successive hydroxylation, is
transformed into serotonin (5-hydroxytryptamine). We re-
ported the occurrence in Citrus genus plants of tryptamine,
serotonin, and all their N-methyl derivatives, that is, N-
methyltryptamine, N,N-dimethyltryptamine, N,N,N-trimethyl-
tryptamine, 5-hydroxy-N-methyltryptamine, 5-hydroxy-N,N-
dimethyltryptamine (bufotenine), and 5-hydroxy-N,N,N-trime-
thyltryptamine (bufotenidine).2 Among these compounds,
N,N,N-trimethyltryptamine and 5-hydroxy-N,N,N-trimethyl-
tryptamine are nicotine-like cholinergic agents by exerting
their action on acetylcholine receptors. 5-Hydroxy-N,N,N-
trimethyltryptamine is a cholinergic agent about 10-fold more
powerful than N,N,N-trimethyltryptamine.4 These pharmaco-
logical properties led us to hypothesize that both compounds
could represent the goal of a biosynthetic pathway targeted to
the defense of citrus plants against aggressors. The second
defensive pathway starts with tyrosine decarboxylation,
Received: October 4, 2016
Revised: January 13, 2017
Accepted: January 16, 2017
© XXXX American Chemical Society
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Figure 1. Structural representations and MS2 transitions utilized for analyses of tyramine derivatives by HPLC-ESI-MS/MS.
Citrus Leaf Extracts. Citrus leaves of sweet orange (Citrus
sinensis), bitter orange (Citrus aurantium), bergamot (Citrus bergamia
Risso & Poit), citron (Citrus medica), lemon (Citrus limon), mandarin
(Citrus reticulata), and pomelo (Citrus maxima) were harvested in
October and December 2014 from the Research Centre for Citrus
Crops and Mediterranean (CRA-ACM), arboretum of Reggio Calabria
(Italy). For each species, 25 g of leaves, finely chopped, was
homogenized in a blender with 100 mL of 0.1% formic acid in
Milli-Q-grade water and then kept under stirring for 30 min. Finally
homogenates were centrifuged at 18000g for 30 min, and supernatants
were stored in 20 mL vials at −20 °C.
Citrus Juice Preparation. The endocarp (the edible part of the
fruit constituted of carpels containing the juice cells) of sweet orange,
bitter orange, bergamot, lemon, and mandarin deprived of seeds was
homogenized in a mixer and then centrifuged at 18000g for 30 min.
Supernatants were stored at −20 °C until used for the subsequent
determinations.
Stapelia hirsuta Extracts. Stapelia hirsuta L. was purchased in a
local plant market. The plant aerial part was used to obtain the
extracts. Slices of the stem (25 g) were homogenized in a blender with
100 mL of 0.1% formic acid in Milli-Q-grade water and then kept
under stirring for 30 min. The homogenate was centrifuged at 18000g
for 30 min and the supernatant stored in 20 mL vials at −20 °C.
HPLC-ESI-MS/MS Analyses. HPLC-ESI-MS/MS analyses were
performed with an HPLC Agilent 1100 series coupled online with an
Agilent LC-MSD SL quadrupole ion trap. MS acquisition was
performed by using electrospray ionization (ESI) in positive-ion
mode. Nitrogen, as both a drying and a nebulizing gas, was used at a
flow rate of 7 L/min and a pressure of 30 psi. The nebulizer
temperature was set at 350 °C. The ion charge control (ICC) was
applied with a target set at 30000 and maximum accumulation time at
20 ms. Measurements were performed from the peak area of the
extracted ion chromatogram (EIC). Multiple reaction monitoring
(MRM) mode was used for detection of analytes. The MS2 transitions
utilized were 138.1 → 121 for tyramine, 152.1 → 121 for N-
methyltyramine, 166.1 → 121 for N,N-dimethyltyramine, 180.1 → 121
for N,N,N-trimethyltyramine, 300.1 → 121 for tyramine-O-β-gluco-
side, 314.2 → 121 for N-methyltyramine-O-β-glucoside, 328.2 → 121
for N,N-dimethyltyramine-O-β-glucoside, and 342.2 → 121 for N,N,N-
trimethyltyramine-O-β-glucoside. HPLC-ESI-MS2 analyses were per-
formed with a Discovery-C8 analytical column (Supelco), 250 mm ×
3.0 mm i.d., 5 μm particle size, eluted isocratically with 0.1% formic
When we analyzed the distribution of methylated tyramine
derivatives in leaves and fruits of several citrus species, we
revealed for the first time the occurrence of N,N,N-
trimethyltyramine, a quaternary ammonium compound,
known as candicine or maltoxin.17 This substance, first detected
in barley rootlets (Hordeum distichon L.),18 acts in animals as a
powerful depolarizing neuromuscular blocker and shows
muscarine-like and sympathomimetic effects.19,20 In this
context, candicine may represent the final step of a biosynthetic
process which, starting from tyramine, is aimed to improve
plant defense.17,21 Recently, we identified a newly metabolic
pathway in Citrus plants leading to the formation of serotonin
5-O-β-glucoside and all its N-methylated derivatives, that is, N-
methylserotonin 5-O-β-glucoside, N,N-dimethylserotonin (bu-
fotenine) 5-O-β-glucoside, and N,N,N-trimethylserotonin
(bufotenidine) 5-O-β-glucoside.22 It is well-known that many
plants store their defense metabolites as inactive glycosides that
can be hydrolyzed by specific glycosidases to release, when
necessary, their toxic defensive aglycons. In this respect, our
results showed that in citrus plants such a glycosylation
mechanism can occur for serotonin and its methylated
derivatives. These recent findings of the occurrence of
glucosylated derivatives of serotonin and all its N-methylated
derivatives in several Citrus genus plants22 prompted us to
investigate the possibility that a similar modification might also
occur for tyramine and its N-methylated derivatives, that is, N-
methyltyramine, N,N-dimethyltyramine (hordenine), and
N,N,N-trimethyltyramine (candicine), whose presence was
previously detected in leaves and fruits of various citrus
plants.17
MATERIALS AND METHODS
■
Reagents. Tyramine, N,N-dimethyltyramine, β-glucosidase from
almonds, and 0.1% solution of formic acid in water were purchased
from Sigma-Aldrich (Milan, Italy). N,N,N-Trimethyltyramine was
synthesized as reported.17 A Discovery-C8 analytical column, 250 × 3.0
mm inner diameter, 5 μm particle size, with guard column of the same
material, was obtained from Supelco (Milan, Italy). All other chemicals
were of analytical reagent grade. Purified water was obtained from a
Milli-Q system from Millipore (Milan, Italy).
B
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Figure 2. Transglucosylation reaction of cellobiose with hordenine catalyzed by almond β-glucosidase.
acid in water, at a constant flow rate of 0.1 mL/min. The injection
track A, panels A1−A4) and coelution with authentic
standards.17 The retention times were 20.5 min for tyramine,
24.0 min for N-methyltyramine, 28.7 min for N,N-dimethyltyr-
amine, and 32.5 min for N,N,N-trimethyltyramine. Interest-
ingly, three more peaks were observed in the chromatogram
(Figure 3, track B). The first (peak B1), at tR 17.2 min,
corresponds to m/z 300.1, the second (peak B2), at tR 19.5
min, corresponds to m/z 314.2, and the third (peak B3), at tR
22.5 min, corresponds to m/z 328.2. All three peaks showed a
common ion fragment at m/z 121 in their MS2 fragmentation
patterns (Figure 3, track B, panels B1−B3), which led us to
suppose that the compounds of peaks B1, B2, and B3 were
structurally related to tyramine, N-methyltyramine, and N,N-
dimethyltyramine, compounds known to form an ion fragment
at m/z 121 in their MS2 fragmentation patterns (Figure 3,
panels A1−A4 from track A). As matter of fact, the MS2
fragment at 121 is generated by neutral loss of ammonia,
methylamine, and N,N-dimethyltyramine, from tyramine, N-
methyltyramine, and N,N-dimethyltyramine, respectively.23 It is
worth noting that neither in bergamot leaf extracts nor in leaf
extracts of other citrus species was the peak at m/z 342.2 with a
MS2 fragment at m/z 121, corresponding to glycosylated
N,N,N-trimethyltyramine, detected.
The common structural motif of the compounds of peaks B1,
B2, and B3 was further supported by the identity of their MS3
fragmentation patterns obtained by isolating the MS2 fragment
at m/z 121 from each of them (Figure 3, panel C). On the
other hand, also the MS3 fragmentation patterns obtained by
isolating the MS2 fragment at m/z 121 of tyramine, N-
methyltyramine, and N,N-dimethyltyramine were identical to
those of the three unknown peaks (Figure 3, panel C).17
Furthermore, all three unknown substances showed in their
MS2 fragmentation patterns ion fragments having mass/charge
ratios coinciding with those of protonated masses of tyramine,
N-methyltyramine, and N,N-dimethyltyramine, that is, m/z
138.1 for peak B1, 152.1 for peak B2, and 166.1 for peak B3
(Figure 3, panels B1−B3 from track B), which likely arise from
the neutral loss of a hexose residue (162 amu) from the parent
ions.23,24 Therefore, taken together, the MS2 fragmentation
patterns of the peaks B1, B2, and B3 led us to reasonably infer
that the unknown compounds were O-glycosylated forms of
tyramine, N-methyltyramine, and N,N-dimethyltyramine, re-
spectively.
volumes were 10−20 μL for each sample or standard solutions.
Enzymatic Synthesis of β-D-Glucosylated Forms of N-
Methylated Tyramine Derivatives. As β-D-glucosides of tyramine
and its N-methylated forms were not commercially available, we
prepared the reference standard substances through a transglucosyla-
tion reaction with cellobiose catalyzed by the β-glucosidase from
almonds. Moreover, only N,N-dimethyltyramine (hordenine) was
commercially available, whereas N-methyltyramine and N,N,N-
trimethyltyramine were not. Therefore, the transglycosylation reaction
was conducted only for hordenine by using the substance to be
glucosylated in pure form (Figure 2). The reaction mixture for the
synthesis of hordenine-O-β-D-glucoside contained 100 mg of
cellobiose, 10 mg of hordenine, and 10 U of β-glucosidase from
almonds in sodium acetate buffer 50 mM at pH 5.0. The reaction was
conducted at 37 °C, and the formation of the product was followed
over time by analyzing 5 μL aliquots of the reaction mixture at 1 h
intervals by HPLC-ESI-MS2 by monitoring the MS2 transition 328.2
→ 121. In order to obtain the glucosylated derivatives of N-
methyltyramine and N,N,N-trimethyltyramine, we first subjected
tyramine to controlled methylation with iodomethane. The reaction
was conducted as previously described,17 and the formation of the
products was monitored over time by HPLC-ESI-MS2. When the
various N-methylated tyramines reached convenient levels, the
methylation reaction was stopped by removing under vacuum the
solvent and excess of iodomethane. Successively, 10−20 mg of the
dried material was subjected to the transglucosylation reaction, as
reported above for hordenine. The formation of products was
monitored by analyzing in MRM mode by HPLC-ESI-MS2 5 μL
aliquots of the reaction mixture every hour through the MS2
transitions 300.1 → 121 for tyramine-O-β-glucoside, 314.2 → 121
for N-methyltyramine-O-β-glucoside, 328.2 → 121 for N,N-
dimethyltyramine-O-β-glucoside, and 342.2 → 121 for N,N,N-
trimethyltyramine-O-β-glucoside.
Statistical Analysis. Data are expressed as mean std of n = 4
determinations, each in duplicate. Differences were assessed by t test,
and a p value less than 0.05 was considered to be significant.
RESULTS AND DISCUSSION
■
Identification and Characterization of the Glycosy-
lated Forms of Tyramine and Its N-Methylated
Derivatives. In the light of our previous findings,17,22 we
investigated whether tyramine and its N-methylated derivatives
(Figure 1) may be subjected to glycosylation in citrus plants as
it happens for serotonin and its N-methylated derivatives.
To this aim, we first subjected leaf extracts of citrus plants to
HPLC-ESI tandem mass analysis. The ESI-MS2 chromatogram
of a bergamot leaf extract was obtained in multiple reaction
monitoring (MRM) mode by isolating the ion masses at m/z
138.1, 152.1, 166.1, 180.1, which coincide with those of the
protonated tyramine, N-methyltyramine, N,N-dimethyltyr-
amine, and N,N,N-trimethyltyramine, and the ion masses at
m/z 300.1, 314.2, 328.2, and 342.2, which coincide with those
of their protonated glycosylated derivatives, respectively
(Figure 3). In the chromatographic conditions employed,
tyramine, N-methyltyramine, N,N-dimethyltyramine, and
N,N,N-trimethyltyramine eluted well resolved. Their identities
were assigned from the MS2 fragmentation patterns (Figure 3,
Mass Spectrometric Characterization of the Glycosy-
lated Forms of Tyramine and Its N-Methylated
Derivatives in Stapelia hirsuta. On the basis of the above
considerations, we sought to investigate the chemical structures
of the putative glycosylated derivatives of tyramine and its N-
methylated forms, which likely occurred in citrus plants. As
such compounds were not commercially available, we took
advantage of a study conducted on Stapelia hirsuta L., a cactus-
like plant belonging to the Apocynaceae family, in which
hordenine, candicine, and hordenine-O-β-D-glucoside were
detected.25 Actually, with the exception of the study of Shabana
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Figure 3. Representative HPLC-ESI-MS2 chromatogram of a bergamot leaf extract. Track A: The chromatogram was obtained in multiple reaction
monitoring (MRM) mode by isolating the ion masses at m/z 138.1, 152.1, 166.1, and 180.1, which coincide with those of the protonated tyramine,
N-methyltyramine, N,N-dimethyltyramine (hordenine), and N,N,N-trimethyltyramine (candicine), respectively, and (track B) by isolating the ion
masses at m/z 300.1, 314.2, 328.2, and 342.2, which coincide with those of their protonated glycosylated derivatives, respectively. Panels A1−A4:
MS2 fragmentation patterns of the peaks in track A. Panels B1−B3: MS2 fragmentation patterns of the peaks in track B. Panel C: MS3 fragmentation
pattern of the MS2 fragment ion (m/z 121.1) common to peaks in tracks A and B. The chromatographic conditions are reported under Materials and
Methods.
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et al.25 and a more recent NMR metabolomic study concerning
the probable occurrence of O-glucosyltyramine in cell cultures
of Cannabis sativa L.,26 as far as we know, the presence of O-
glycosylated derivatives of tyramine or its N-methylated
derivatives in other sources has not been described. Therefore,
with the aim to obtain at least one reference standard to
compare with the unknown substances in citrus plants, extracts
from the aerial parts of Stapelia hirsuta were subjected to mass
spectrometric characterization. Also in this case, the chromato-
gram (Figure 4) was obtained in multiple reaction monitoring
(MRM) mode by isolating the ion masses at m/z 138.1, 152.1,
166.1, and 180.1, which coincide with those of the protonated
tyramine, N-methyltyramine, N,N-dimethyltyramine, and
N,N,N-trimethyltyramine, and the ion masses at m/z 300.1,
314.2, 328.2, and 342.2, which coincide with those of their
protonated glycosylated derivatives, respectively. Results
showed the occurrence of hordenine and candicine in the
extract of Stapelia hirsuta (Figure 4, track A), thus confirming
what was previously reported.25 However, two intense peaks
corresponding to tyramine and N-methyltyramine were
detected. The identity of these compounds, not previously
mentioned,25 was confirmed by comparison with authentic
standards. As for glycosyltyramine derivatives, four well
resolved peaks were observed in the chromatogram (Figure 4,
track B). The first (peak B1), at tR 17.2 min, corresponds to ion
mass of m/z 300.1, the second (peak B2), at tR 19.5 min,
corresponds to ion mass of m/z 314.2, the third (peak B3), at tR
22.5 min, corresponds to ion mass of m/z 328.2, and the fourth
(peak B4), at tR 26 min, corresponds to ion mass of m/z 342.2.
The peak B3 at tR 22.5 min, isolated at m/z 318.2, shows a MS2
fragmentation pattern (Figure 3, panel B3) which confirms the
previously reported presence of hordenine-O-β-D-glucoside in
the Stapelia extract.25 In fact, the MS2 fragment at m/z 166
(corresponding to protonated hordenine) is generated by the
neutral loss of the glucosyl residue from the protonated
compound, while the MS2 fragment at m/z 121 arises from the
dimethylamine neutral loss (45 amu) from the fragment at m/z
166. Furthermore, the MS3 analysis conducted by isolating the
MS2 fragment at m/z 121 showed the same fragmentation
pattern as that obtained by isolating the fragments at m/z 121
which arise from MS2 fragmentation of tyramine and its N-
methyl derivatives (data not shown). As for peaks B1 and B2
(Figure 4, track B), similar considerations to those reported
above reveal their possibly identities as tyramine-O-β-D-
glucoside (peak B1) and N-methyltyramine-O-β-D-glucoside
(peak B2), so far not reported in this plant. Notably, peaks B1
and B2 showed the same retention times and MS2
fragmentation pattern as peaks B1 and B2 found in the extracts
of citrus plants (Figure 3, panels B1−B3 from track B). It is also
interesting to note the peak B4 at m/z 342.2, whose MS2
fragmentation pattern is consistent with that of N,N,N-
trimethyltyramine-O-β-D-glucoside (Figure 4, panel B4). In
fact, the MS2 fragment at m/z 180 (corresponding to m/z of
N,N,N-trimethyltyramine) is generated by the neutral loss of
the glucosyl residue from the parent ion, while the MS2
fragment at m/z 121 arises from the trimethylamine neutral
loss (59 amu) from the fragment at m/z 180. This was further
substantiated by the MS3 fragmentation pattern obtained by
isolating the MS2 fragment at m/z 180, where, besides the MS3
fragment at m/z 121, also present is a fragment at m/z 60
corresponding to the trimethylammonium ion.
Figure 4. Representative HPLC-ESI-MS2 chromatogram of a Stapelia
hirsuta extract. Track A: The occurrence of tyramine and all its N-
methylated derivatives (peaks A1−A4) was confirmed by comparison
with authentic standards. Track B: MS2 fragmentation patterns of the
peaks B1 (tR 17.2 min, m/z 300.1), B2 (tR 19.5 min, m/z 314.2), B3
(at tR 22.5 min, m/z 328.2), and B4 (at tR 26 min, m/z 342.2). The
chromatographic conditions are reported under Materials and
Methods.
Derivatives Produced by Enzymatic Synthesis. The
identity of peak B3 in the chromatogram of the bergamot
leaf extract (Figure 3) as hordenine-O-β-D-glucoside was
ascertained by its retention time and MS2 fragmentation
pattern, which were the same as those of peak B3 in the
chromatogram of the Stapelia extract (Figure 4, track B), where
the occurrence of hordenine-O-β-D-glucoside was known.25
Moreover, peaks B1 and B2 in the Stapelia extract chromato-
gram (Figure 4, track B) show the same retention times and
Mass Spectrometric Characterization of the Glucosy-
lated Forms of Tyramine and Its N-Methylated
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MS2 fragmentation patterns as peaks B1 and B2 of the
bergamot leaf extract (Figure 3, track B) which, on the basis of
their MS2 fragmentation patterns, led us to suppose that they
were tyramine-β-D-glucoside and N-methyltyramine-β-D-gluco-
side. Furthermore, peak B4 (Figure 4, panel B) showed a MS2
fragmentation pattern which was consistent with its identity as
N,N,N-trimethyltyramine-O-β-D-glucoside. Indeed, we did not
observe a peak analogous to peak B4 in any citrus plant extract.
In order to confirm the identities of these three substances in
Stapelia, we performed their enzymatic synthesis by a
transglycosylation reaction with cellobiose catalyzed by β-
glucosidase from almonds. The HPLC-ESI-MS2 time course of
the transglycosylation reaction showed that all the expected
glucosides of tyramine and its N-methylated derivatives were
formed and showed the same chromatographic retention times
and MS2 fragmentation patterns as the corresponding peaks in
the chromatograms of the extracts from citrus plants and
Stapelia (Figure 5).
Figure 5. Time course of transglucosylation reaction catalyzed by
almond β-glucosidase. Transglucosylation reaction mixture, besides
cellobiose, tyramine, and enzyme, contained N-methyltyramine, N,N-
dimethyltyramine, and N,N,N-trimethyltyramine previously produced
by controlled methylation of tyramine with iodomethane (see
Materials and Methods). The MS2 extracted ion chromatograms in
MRM mode, obtained by isolating the ion masses at m/z 300.1, 314.2,
328.2, and 342.2, show the production over time of glucosylated
tyramine and its N-methylated derivatives, respectively. The m/z of
the parent and extracted ions are the same as on the corresponding
peaks in Figure 4, track B. (A) 2 h, (B) 4 h, and (C) 8 h of incubation.
The four peaks showed the same chromatographic retention times and
MS2 fragmentation patterns as the corresponding peaks in the
chromatograms of the extracts from citrus plants and Stapelia hirsuta.
Figure 6. Relative levels of glucosylated tyramine and its N-methylated
derivatives in various Citrus species. Graphs are normalized to 100%
representing the level of the specific compound in the top productive
species. Panel A: tyramine-O-β-D-glucoside. Panel B: N-methyltyr-
amine-O-β-D-glucoside. Panel C: N,N-dimethyltyramine-O-β-D-gluco-
side. The left side of each panel shows relative levels of the specific
compound in leaves, the right side, in juice. The level of each
compound is the average of four determinations. Bars represent the
standard deviations.
Distribution of Tyramine Glucosylated Derivatives in
Citrus Plants. The levels of the glucosylated tyramine and its
N-methylated derivatives were determined in leaves and juices
of sweet and bitter orange, bergamot, mandarin, lemon, citron,
and pomelo (Figure 6, panels A−C). For each citrus species,
the levels of the glucosylated tyramine and its N-methylated
derivatives are normalized to 100% for the plant species that
showed the highest level of the specific metabolite (Figure 6,
panels A−C). It appears evident that leaves are the tissue part
of plants where these substances accumulate mostly. In juices,
the basal levels of these substances were very low or even zero,
as in the case of lemon juice (Figure 6, right sides of panels A−
C). The glucosylated tyramine derivatives reached the highest
levels in sweet and bitter orange leaves, whereas such levels are
mainly low in all other species (Figure 6, panels A−C). A
noticeable diversity between citrus plants and Stapelia consists
in the fact that, although N,N,N-trimethyltyramine (candicine)
has been detected in leaves of citrus plants,17 its glucosylated
derivative was not observed in any of them, whereas, as seen
above, in the Stapelia extracts it occurs together with all other
glucosylated tyramine derivatives. This observation suggests the
absence in citrus plant genus of the biosynthetic machinery for
the two possible pathways that may produce this substance, that
is, N-methylation of hordenine-1-O-β-D-glucoside or glucosyla-
tion of candicine (Figure 7).
Interrelationship of the Tyramine Derivatives in Citrus
Genus. In this paper we show for the first time the presence of
glucosylated forms of tyramine and its N-methylated derivatives
in Citrus genus plants. Noticeably, these results highlight the
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Figure 7. Scheme of the putative metabolic pathways for the biosynthesis of the tyramine derivatives in citrus plants. First step of the pathways is the
formation of tyramine from tyrosine, catalyzed by the enzyme tyrosine decarboxylase (TYDC). Successively, N-methylation of tyramine is probably
accomplished by the action of the same or different methyltransferase(s) acting in consecutive manner. Then, each methylated intermediate could be
transformed in the corresponding glucoside by reactions likely catalyzed by UDP glucosyltransferase(s), which transfer the sugar residue from UDP-
glucose, acting as a donor molecule. Candicine is reported to occur in citrus plants, but the corresponding glucoside was not detected. This is likely
due to the absence of the biosynthetic machinery for the two possible pathways that may produce this substance, that is, N-methylation of
hordenine-1-O-β-D-glucoside or glucosylation of candicine. Octopamine originates by hydroxylation of tyramine catalyzed by tyramine β-hydroxylase
(TBH). It is worth noting that, in citrus plants, the adrenergic amine synephrine (N-methyloctopamine) is not produced by methylation of
octopamine, as in animals, but by hydroxylation of N-methyltyramine.
parallelism between the metabolic pathways of tyramine and
serotonin in this plant genus. Indeed, in our previous studies we
showed the occurrence of serotonin (5-hydroxytryptamine)
and all its N-methylated derivatives2,3 and, successively, also
that of their glucosylated forms.22 Likewise, tyramine and its N-
methylated derivatives (N-methyltyramine, N,N-dimethyltyr-
amine, and N,N,N-trimethyltyramine) were found to occur in
citrus plants17 and, as here reported, also the glucosylated
derivatives of tyramine, N-methyltyramine, and N,N-dimethyl-
tyramine but not the glucosylated derivative of N,N,N-
trimethyltyramine. As for the biosynthesis of those compounds,
at present, there are no specific studies on the subject.
However, we hypothesize that a putative metabolic pathway
may comprise two steps of methylation and glycosylation of
tyramine, involving a sequence of reactions starting with the
formation of tyramine from tyrosine, catalyzed by the enzyme
tyrosine decarboxylase (TYDC) (Figure 7). Afterward, tyr-
amine might be N-methylated by the action of the same or
different methyltransferase(s) in consecutive steps (Figure 7).
The pathway could end by the transfer of the sugar residue to
tyramine and its N-methylated derivatives. Alternatively,
tyramine-O-β-D-glucoside could be N-methylated to produce
in a consecutive manner the forms at higher methylation degree
(Figure 7).
serotonin, accumulate in plants in response to the infection by
fungal pathogen.32 It is well-known that glycosylation, by
making many compounds less reactive and more water-soluble,
is a way to prevent cell damage and safely store toxic
compounds employed by plants for defensive aims. We found a
greater presence of the glucosylated tyramine derivatives in
leaves than in fruits (Figure 6, panels A−C), which supports the
proposed role of glycosides in plant defense.33−36 In fact, leaves
are the part of plants more susceptible to aggression by
herbivores and pathogens and, for this reason, they require
effective means of defense. As known, when leafy tissues are
damaged by attackers, the glycosides, compartmentalized
mostly in the vacuoles, undergo the action of specific
glycosidases, which can occur either in the plant or also in
the aggressors’ tissues.35,36 Consequently, the aglycons are
released and can play their deterrence and toxicity roles.35,36 In
this respect, the coexistence in citrus plants of tyramine and
serotonin derivatives suggests the common finality of all these
secondary metabolites to the defensive response against biotic
stress in this important plant genus.
AUTHOR INFORMATION
■
Corresponding Author
The distribution of N-methylated derivatives of tyramine17
and its glycosylated forms resembles that of other Citrus
metabolites involved in defense and resistance mecha-
nisms,27−30 such as limonoids and flavanones, which act as
powerful natural pesticides31 and antifungal agents.27,30
From a physiological point of view, the biosynthesis of the
glucosylated metabolites could be likely linked to the role
played in the plant defense by their aglycons, which affect
processes essential for plant aggressors, such as fertility,
locomotion, spawning, and hatching rate.8 Some aglycons
even act as antimicrobial metabolites which, as in the case of
*Phone: +39-081-5665865. Fax: +39-081-5665863. E-mail:
luigi.servillo@unina2.it.
ORCID
Luigi Servillo: 0000-0002-9728-522X
Funding
This research was supported by Progetto PON03PE_00060_2
(DD 713/2010).
Notes
The authors declare no competing financial interest.
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(22) Servillo, L.; Giovane, A.; Casale, R.; D’Onofrio, N.; Ferrari, G.;
Cautela, D.; Balestrieri, M. L.; Castaldo, D. Serotonin 5-O-β-glucoside
and its N-methylated forms in citrus genus plants. J. Agric. Food Chem.
2015, 63, 4220−4227.
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