Methyl Jasmonate Stimulates Biosynthesis of 2-phenylethylamine, Phenylacetic Acid and 2-phenylethanol in Seedlings of Common Buckwheat

Methyl jasmonate has a strong effect on secondary me-tabolizm in plants, by stimulating the biosynthesis a number of phenolic compounds and alkaloids. Common buckwheat (Fagopyrum esculentum Moench) is an important source of biologically active compounds. This research focuses on the detection and quantification of 2-phenylethylamine and its possible metabolites in the cotyledons, hypocotyl and roots of common buckwheat seedlings treated with methyl jasmonate. In cotyledons of buckwheat sprouts, only traces of 2-phenylethylamine were found, while in the hypocotyl and roots its concentration was about 150 and 1000-times higher, respectively. Treatment with methyl jasmonate resulted in a 4-fold increase of the 2-phenylethylamine level in the coty-ledons of 7-day buckwheat seedlings, and an 11-fold and 5-fold increase in hypocotyl and roots, respectively. Methyl jasmonate treatment led also to about 4-fold increase of phenylacetic acid content in all examined seedling organs, but did not affect the 2-phenylethanol level in cotyledons, and slightly enhanced in hypocotyl and roots. It has been suggested that 2-phenylethylamine is a substrate for the biosynthesis of phenylacetic acid and 2-phenylethanol, as well as cinnamoyl 2-phenethylamide. In organs of buckwheat seedling treated with methyl jas-monate, higher amounts of aromatic amino acid trans-aminase mRNA were found. The enzyme can be involved in the synthesis of phenylpyruvic acid, but the presence of this compound could not be confirmed in any of the examined organs of common buckwheat seedling.

2-Phenylethylamine (PEA) rarely occurs in plants, and its physiological role remains unclear (Smith, 1977;Shabana et al., 2006).It is product of l-phenylalanine (l-PHE) decarboxylation by aromatic l-amino acid decarboxylase (Smith, 1977).PEA often serves as an indicator of food quality and freshness, since it can be synthesized by fungi and bacteria contaminating the foodstuffs (Onal et al., 2013).In tomato fruit, PEA can be transformed into phenylacetaldehyde (PAAld), which is further converted to 2-phenylethanol (PE), an important component of tomato aroma (Tieman et al., 2006;2007).PE is also a major constituent of rose flower scent (Sakai et al., 2007), and various fruits (Aubert et al., 2005).In rose flowers, PE is biosynthesized from l-PHE via PAAld by aromatic amino acid decarboxylase (AADC) and phenylacetaldehyde reductase (Sakai et al., 2007).In protoplast isolated from rose petals, aromatic amino acid aminotransferase transforms l-PHE to phenylpyruvic acid, which is then converted to PAAld and subsequently to PE (Hirata et al., 2012).PAAld, an important component of aroma, has also been found in buckwheat seeds (Janeš et al., 2009).Moreover, PE and PAAld have been detected among volatile flavor compounds of boiled buckwheat flour (Yajima et al., 1983).
Biosynthesis of phenylacetic acid (PAA) in plants probably occurs with the participation of aromatic aminoacid transaminase (AAT) which can produce phenylpyruvate from the l-PHE (Gamborg &Wetter, 1963;Tomè et al., 1975).Then, decarboxylation and subsequent oxidation of phenylpyruvate leads to PAAld and PAA.PAA was identified as a natural auxin-like growth regulator in plants, but its full physiological role is not clear (Wightman & Lighty, 1982;Morris & Johnson, 1987;Ludwig-Müller & Cohen, 2002).
Common buckwheat is a dicotyledonous plant from Polygonaceae family.Buckwheat sprouts are a rich source of flavonoids, and other phenolic compounds (Kim et al., 2004;2011).Flavonoids are derived from l-PHE, and PEA is also synthesized from this amino acid.Enormous accumulation of PEA in buckwheat seedlings treated with MJ could have an impact on the synthesis of flavonoids.It seems that such substrate competition may be in buckwheat hypocotyl.In this buckwheat organ MJ inhibits the synthesis of anthocyanins, and stimulates the production of PEA (Horbowicz et al., 2011b).On the other hand, is not fully understood the reason for the accumulation of PEA, as well as its further metabolic transformation.It is likely that PEA can be a substrate in the formation of PAA, PAAld and PE in buckwheat.This study focuses on the detection and quantification of PEA and its possible metabolites in organs of common buckwheat seedlings treated with MJ.An additional objective has been to conduct genetic tests describing the biosynthesis of PEA and its metabolites.

MATERIAL AND METHODS
Preparation of plant samples.Seedlings of common buckwheat (cv.Hruszowska) were prepared by germination of seeds between two layers of wet filter paper which were then rolled and inserted in a 2.5 L beaker containing about 200 cm 3 of tap water.The germination proceeded in darkness at 24±1°C.After four days the seedlings were transferred to a growth chamber at 22±2°C/18±2°C (day/night: 16 h/8 h).Light (100 μmol×m -2 ×s -1 ) was provided by high-pressure sodium lamps.Then, the half of seedlings were treated with atmospheric vapors of methyl jasmonate (MJ, Sigma Aldrich).A 3-cm-wide ribbon of filter paper containing a drop of MJ (56 mg) was placed against the inner wall of the beaker containing the rolls with the seedlings, and the jar was immediately closed tightly with a transparent silicon film.The concentration of atmospheric MJ was calculated a 0.1 mM (assuming its complete evaporation).Immediately after germination, and after four or seven days of growth in such conditions, organs (roots, hypocotyl and cotyledons) of control plants and MJ treated were subjected to analyses.The seedlings were taken for analyses between 9 and 10 am.For molecular analyses the plant samples were lyophilized for a period of 72 h in a freeze dryer Christ Alpha 1-2 LD+.For genetic tests after 4 days of MJ treatment the seedling organs have been taken, which was then stored until analysis at -85°C.
Analysis of metabolites by HPLC-MS/MS.About 0.05 g of freeze-dried and pulverized samples of cotyledons, hypocotyl and roots of buckwheat was extracted with 2 mL of 80% methanol by 60-s sonication (VC 750, Sonics & Materials, USA) and 30-s vortexing.Subsequently, the extraction was conducted using Thermomixercomfor (Eppendorf) by shaking at 20°C with amplitude of 600 rpm for 24 h.After centrifugation (20 min, 13000×g, 4°C), the obtained supernatant was stored at -80°C until analysis.Analysis of 2-phenylethylamine (PEA), 2-phenylethanol (PE) and phenylacetic acid (PAA) was carried out by using HPLC-MS/MS system consisting of two pumps (LC-20AD), autosampler (SIL-20AC HT ), column oven (CTO-10 AS VP ), degasser (DGU-20A 3 ), system controller (SCL-10 A VP ) and QTRAP 5500 mass spectrometer (AB SCIEX, Canada) with a triple quadrupole, an ion trap, and an ion source of electrospray ionization.A 5-mL aliquot of the extract was injected to the HPLC-MS/MS system equipped with a 150×2.1 mm i.d.XBridge C18, 3.5 µm column (Waters, Milford, USA).Chromatography was performed at 45°C with a flow rate of 0.23 mL×min -1 and a step gradient system of water/formic acid (99:1, v/v, phase A) and acetonitrile/formic acid (99:1, v/v, phase B) comprising 10-80-80-10-10% B at 0-10-15-16-35 min.Qualitative analysis consisted of, among others, scanning in a positive and negative ion mode.Scanning fragment ions formed from the decay of the parent ion was also performed.This method was used in the search for presence of fragmentation ions of phenylpyruvate.The identity of PEA, PE, PAA and PAAld was confirmed by comparing the HPLC retention times and MS/MS spectra to the ones obtained for authentic standards (Sigma Aldrich).Quantitative analysis was performed by means of the MRM (Multiple Reaction Monitoring) method using positive ionization for: 2-phenylethylamine (122-105 m/z), phenylacetaldehyde (121-91 m/z), and an unknown compound (probably cinnamoyl 2-phenylethylamide, 251-77 m/z).
Measurement of aromatic amino acid transaminase gene expression.Total RNA was isolated from the organs of buckwheat seedlings with the Spectrum Plant Total RNA Kit (Sigma Aldrich), and residual genomic DNA was removed using On-Column DNase I Digestion Set (Sigma Aldrich, Poland).Qualitative and quantitative analyses of the RNA were performed using an Epoch UV-Vis microplate spectrophotometer.Subsequently, the intact total RNA (1 µg) was used for reverse transcription using the High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Life Technologies, Poland).Negative controls (no template and no reverse transcriptase) were included.The mRNA level of the aromatic amino acid transaminase (AAT) gene in MJ-treated and control seedlings of F. esculentum was assessed with the use of quantitative real-time polymerase chain reaction (RT-qPCR).Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was selected as the reference gene.The mRNA level of the GAPDH gene was quantified using Custom TaqMan Gene Expression Assay (Life Technologies, Poland); the following primers and TaqMan fluorescent probe were used: forward primer: TGGAGCTGCTAAGGCTGTTG, reverse primer: GCCATTCCAGTCAACTTTCCATT, and probe: FAM-CAACACTGGCAACACC-NFQ.The relative expression of the AAT gene was quantified using the TaqMan Gene Expression Assay (no.At02227046_g1, Life Technologies).The reaction mixture consisted of 4 µl of cDNA, 1 µl 20× TaqMan Gene Expression Assay, 10 µl 2× TaqMan Fast Universal PCR Master Mix, and 5 µl RNase-free (PCR grade) water.Measurement of transcript amounts was performed using the fast mode of StepOne Plus Real-Time PCR System and StepOnePlus Software v2.3 (Life Technologies, USA).Quantification of the relative level of AAT mRNA was carried out according to the ΔΔC T method described by Livak & Schmittgen (2001).The obtained data are expressed as average n-fold changes ± standard deviation (S.D.) in levels of AAT transcript in comparison to untreated plants.The experiments were performed with three independent biological replicates.
Statistical analysis.The analyses of PEA, PE and PAA levels were determined in three technical replicates, and the results are presented as means ± standard deviation (S.D.).The differences between the contents of analyzed compounds in the treated and control seedlings were analyzed statistically using Student's t-test, P<0.01.
One-way analysis of variance (ANOVA) with Tukey's post hoc test was used to evaluate the significance of differences of the AAT gene transcript between MJtreated and control seedlings (P<0.05 was considered significant).Statistical calculations were conducted using STATISTICA 10 software (StatSoft, Poland).

RESULTS
HPLC/MS/MS analysis showed the presence of 2-phenylethylamine (PEA), 2-phenylethanol (PE) and phenylacetic acid (PAA) in the cotyledons, hypocotyl and roots of buckwheat seedlings, as confirmed by comparing their fragmentation spectra and retention times to the obtained commercial standards.However, this analysis did not confirm the presence of phenylacetaldehyde (PAAld) in the examined organs.
Etiolated buckwheat seedlings obtained after 4-day germination in darkness have light-yellow cotyledons, 5-7 cm tall white hypocotyl and a root system with the primary root, 8-10 cm long.In cotyledons of such seedlings, a very low level of PEA was found (Fig. 1).In hypocotyl, its concentration was about 150 times higher than in cotyledons, and in roots about 1000 times.During the first four days of de-etiolation, the level of PEA increased in cotyledons and decreased in hypocotyl and roots.After seven days growth in the light conditions, the content of PEA increased in the all organs, and was almost equal in cotyledons and roots, and slightly lower in hypocotyl (Fig. 1).
Methyl jasmonate (MJ) caused enormous increase of the PEA level in all the examined tissues of buckwheat seedlings in comparison with control plants at the same stage.It was particularly spectacular after 4 days of treatment with MJ, when PEA reached the highest level in roots and a slightly lower, but still a high one in hypocotyl and cotyledons.After seven days of MJ action the PEA level in cotyledons was 4-fold higher, 11-fold higher in hypocotyl, and 5-fold in roots compared to control (Fig. 1).
The PAA content remained constant throughout of de-etiolation process in cotyledons and hypocotyl of seedlings (Fig. 2).In roots, it rose only slightly during the first four days, and then increased 5-fold at 7 days of de-etiolation.A four-day use of MJ led to a 3-fold increase of PAA content in cotyledons and roots, and a about 7-fold in the hypocotyl.After seven-days use of MJ action a further accumulation of PAA was observed, to a level exceeding 5, 9 and 4-fold its content in cotyledons, hypocotyl and roots of control plants, respectively (Fig. 2).
2-Phenylethanol (PE) was present in the organs studied at much lower (ng) concentrations than PEA and PAA (µg) (Fig. 3).In cotyledons and hypocotyl its level remained relatively stable during 4 or 7 days of de-etiolation, and rose gradually in roots.
The use of MJ (for 4 or 7 days) did not affect the content of the PE in cotyledons, but significant increase occurred in the hypocotyl after 4 days, and in roots after 7 days of MJ treatment.
To study the effect of MJ on the expression of genes encoding enzymes involved in the metabolism of l-phenylalanine to PEA, PAA, PAAld and PE, we attempted to quantitate mRNA levels of l-phenylalanine decarboxylase, alcohol dehydrogenase, aldehyde oxidase 1 and 2, aromatic aldehyde synthase, copper amine oxidase 1, lipoxygenase and aromatic acid transferase (AAT).Since no nucleotide sequences for these genes from F. esculentum in GenBank or other databases are available, we selected homologous genes from other plants related to the Fagopyrum genus (e.g.Polygonum minus Huds., Arabidopsis thaliana L.).However, Custom TaqMan Gene Expression Assays (Life Technologies, Poland) targeting were ineffective with cDNA from Fagopyrum esculentum.Presumably, the prepared primers and/or TaqMan probes were not sufficiently complementary to the buckwheat cDNA templates.The only exception was AAT gene, for which specific amplification was achieved.
Table 1 shows the results of the AAT mRNA quantitation in diverse organs of seedlings subjected to MJ The results for control and MJ-treated plants were significantly different (P<0.01): for cotyledons, hypocotyl, and roots (4-days and 7-days treated).Seedlings prepared within 4-days sprouting in the darkness were transferred to the growth chamber and then treated with atmospheric MJ for 4-or 7-days.Chemical analyzes have been performed immediately after germination, and after 4 and 7 days of treatment.
(0.1 mM vapour) for four days compared to untreated seedlings.
In all the organs studied MJ enhanced the level of AAT mRNA.The highest (7.50-fold) was noted in roots, a moderate (5.22-fold) increase was in the hypocotyl, and the lowest (3.60-fold) in cotyledons.A oneway analysis of variance with Tukey's post hoc test confirmed the statistical significance of the effect of MJ (D f =2; F (2,6) =6.467; P<0.0001).

DISCUSSION
A much lower content (traces) of PEA was found in cotyledons then in roots and the hypocotyl of etiolated buckwheat seedlings, which could indicate that PEA is synthesized in the roots in that phase of seedling growth, and then transported to other tissues during further development.Following exposure to light, as after 4 and 7 days of growth the content of PEA reached a similar level in all the examined organs.Such a phenomenon has been described for the first time.Remains, however, to determine whether it is the effect of light or plant growth.Also the differences between responses of organs studied to MJ were statistically significant.
Because of the scarce data on the role of PEA in plants, the finding remains just an assumption on this subject.There is no known reason for the accumulation of PEA and its possibly transport observed in young buckwheat seedlings.Smith (1977) formulated a hy- The results for control and MJ-treated plants were significantly different (P<0.01): for cotyledons and hypocotyls (4-and 7-days treated); for roots (7-days treated) .For roots (4-days treated) the differences were not significant.Seedlings prepared within 4-days sprouting in the darkness were transferred to the growth chamber and then treated with atmospheric MJ for 4-or 7-days.Chemical analyzes have been performed immediately after germination, and after 4 and 7 days of treatment.

Figure 3. Effect of de-etiolation and MJ on 2-phenylethanol content in organs of common buckwheat seedling.
The results for control and MJ-treated plants were significantly different (P<0.01): for hypocotyls, 4-days treated; for roots, 7-days treated.For cotyledons (4-and 7-days treated), for roots (4-days treated) and for hypocotyls (7-days treated) the differences were not significant.Seedlings prepared within 4-days sprouting in the darkness were transferred to the growth chamber and then treated with atmospheric MJ for 4-or 7-days.Chemical analyzes have been performed immediately after germination, and after 4 and 7 days of treatment.

Table 1. Effect of MJ treatment on relative expression of AAT gene in the seedlings of common buckwheat.
Seedlings prepared within 4-days sprouting in the darkness were transferred to the growth chamber and then treated with atmospheric MJ for 4-days.The data are presented as the mean AAT amount ± S.D. Analysis of gene expression was performed in three biological and three technical replicates.Different letters denote significant differences between organs of treated seedlings and control at P<0.05.pothesis that plants may accumulate PEA as part of a defense system against insects.Another reason for the PEA presence could be its allelopathic properties (Szwed et al., 2014).It is known that buckwheat extracts suppress some weeds (Iqbal et al., 2005) and inhibit growth of wheat and lettuce seedlings (Mioduszewska et al., 2013).One of the postulated routes of the metabolism of PEA in plants is its oxidation by monoamine oxidase (MAO).Indeed, a high specificity of MAO for PEA has been found in seedlings of Avena sativa (Zhang et al., 2012).MAO can oxidise PEA to PAAld, and a further reduction of PAAld to PE, catalyzed by phenylacetaldehyde reductase, has been described in tomato (Tieman et al., 2006;2007) and in rose tissues (Sakai et al., 2007;Chen et al., 2011).However, the enzyme that oxidizes PAAld to PAA has not been characterized in plants yet.On the other hand, according to Ludwig-Müller & Cohen (2002), the presence of nitrilase enzyme in nasturtium suggests that the biosynthesis of PAA uses the nitrilase pathway with a benzylglucosinolate precursor.Nitrilases are enzymes found in many plants, which convert nitriles to the corresponding carboxylic acids.For example, indole3-acetic acid (IAA) can be synthesised through the indole-3-pyruvic acid pathway or the tryptamine pathway (Mano & Nemoto, 2012).The tryptamine pathway involves conversion of tryptophane into tryptamine, and then to indole-3-acetonitrile, which is further converted by a nitrilase into IAA.Nitrilases of Sorghum bicolor exhibited a high enzymatic activity towards 4-hydroxyphenyl-acetonitrile and phenylacetonitrile, and converted these compounds into hydroxyphenylacetic acid and PAA, respectively (Jenrich et al., 2007).It is possible that the latter mentioned metabolic pathway functions in the common buckwheat, although this requires further study.
PAA has a much weaker auxin activity than IAA (Wightman & Lighty, 1982).However, the transport of PAA competes with IAA translocation, and may therefore affect the plant growth (Morris & Johnson, 1987).An increased level of PAA as a result of MJ treatment in buckwheat organs, and consequently a reduced IAA transport, could be the major reason for the inhibition of root growth reported in our previous studies (Horbowicz et al., 2008).It is also speculated that PAA preferentially plays a role in the root interactions with soil microorganisms, which could explain its elevated level in roots of buckwheat seedlings (Morris & Johnson, 1987).PAA has also some allelopathic activity (Szwed et al., 2014).
The role of PE in buckwheat seedlings is unknown.It could be a substrate for the biosynthesis of phenylacetaldehyde (PAAld), whose presence has been found in the scent of buckwheat seeds (Janeš et al., 2009).The presence of both PE and PAAld has also been confirmed in the flavor of boiled buckwheat flour (Yajima et al., 1983).According to our recent studies, PE has some allelopathic properties, because it inhibits of seedlings growth of several plant species (Szwed et al., 2014).
Searching for other possible route of PEA transformation in buckwheat seedlings we therefore undertook attempts to find another PEA metabolites.According to our previous studies trans-cinnamic acid is the major phenolic acid in organs of buckwheat seedling (Horbowicz et al., 2011a).Therefore, we searched for a compound which is a derivative of PEA and trans-cinnamic acid, cinnamoyl 2-phenylethylamide (C 6 H 5 -CH 2 -CH 2 NH-CO-CH=CH-C 6 H 5 ) could be such compound.It is an amide in which trans-cinnamic acid provides the acid moiety and 2-phenethylamine the amine moiety.In the mass spectra an ion at m/z 251.1376 (calculated mass: 251.1311) was observed.The ion at m/z 251 (M + ;17) was fragmented to one at m/z 234 which was attributed to the loss of NH 3 (M + -NH 3 ; 49).The support for the phenylethyl moiety come from an intense peak (100, base peak) at m/z 105, likely corresponding to C 6 H 5 -CH 2 -CH 2 + .Such a base peak (m/z 105) has been observed also in spectra of N-phenylethyl-dodecanamide, N-phenylethyl-tridecanamide and N-(2-phenylethyl)-indomethacin amide (Metlin Scripps Database).Other peaks noted in our mass spectrum were the following m/z: 77 (C 6 H 5 + ; 6); 84 (45); 122 (C 6 H 5 -CH 2 -CH 2 -NH 3 + ; 34) and 130.The peak at m/z 130 (probably C 6 H 5 -CH=C-CO + ; 6) likely comes from the cinnamoyl part of the amide.The peak at m/z 205 (M -46) is probably the result of McLafferty rearrangement.Thus, we suppose that PEA can be a substrate for the synthesis of cinnamoyl 2-phenylethylamide.However, a quantitative analysis of this compound was not possible due to a lack of a pure standard.
The positive linear relationship (r=0.601,P<0.05) between PAA and PEA content, and between PAA and PE (r=0.677,P<0.01) suggests their common biosynthetic pathway.Similarly, a positive linear relationship was also observed between the levels of PEA and cinnamoyl 2-phenylethylamide (r=0.611,P<0.05).In contrast no significant correlation between PE and PEA was found.The explanation for this situation is difficult.It is known that secondary metabolism in plants is complicated and regulated by a complex set of genes, encoding diverse regulators, modifiers, transporters, and enzymes responsible for the synthesis and release of elicitors (Kim et al., 2011).
The results of our study show that all the MJ-treated organs of buckwheat seedlings contained increased amounts of amino acid transaminases (AAT) mRNA.These results are in line with the suggestion that aromatic amino acid transaminases may be involved in the production of PE in tomato plants (Tieman et al., 2006) and also confirm previous studies on tyrosine amino acid transferase (TAT).Recently, Zhang et al. (2014) reported that MJ treatment significantly induced the transcriptional activity of TAT gene in hairy roots of Salvia miltiorrhiza.Earlier, a similar phenomenon was observed for TAT in a jasmonate-deficient mutant of Arabidopsis thaliana (Sandorf & Holländer-Czytko, 2002).
The results obtained in our study suggest that the biosynthesis of PAA and PE in buckwheat tissues likely proceeds through phenylpyruvate, which can be obtained by the action of AAT.However, no signals confirming the presence of phenylpyruvate could be found in the mass spectra obtained here by LC-MS/MS method.

CONCLUSIONS
The obtained results do not allow us to conclude definitely as to the identity of biosynthetic pathway of phenylacetic acid and 2-phenylethanol.It seems that both routes are possible: by the deamination of l-phenylalanine to phenylpyruvic acid and/or by the oxidation of 2-phenylethylamine by a multifunctional enzyme or a set of enzymes.Further studies, particularly molecular ones, are necessary to resolve this problem.The occurrence of phenylacetic acid, 2-phenylethanol, and probably cinnamoyl 2-phenylethylamide in roots, hypocotyl and cotyledons of common buckwheat seedlings was confirmed for the first time.Their biosynthesis is stimulated by methyl jasmonate.

Figure 1 .
Figure 1.Effect of de-etiolation and methyl jasmonate (MJ) on 2-phenylethylamine (PEA) content in organs of common buckwheat seedling.The results for control and MJ-treated plants were significantly different (P<0.01): for cotyledons, hypocotyl, and roots (4-days and 7-days treated).Seedlings prepared within 4-days sprouting in the darkness were transferred to the growth chamber and then treated with atmospheric MJ for 4-or 7-days.Chemical analyzes have been performed immediately after germination, and after 4 and 7 days of treatment.

Figure 2 .
Figure 2. Effect of de-etiolation and methyl jasmonate (MJ) on phenylacetic acid (PAA) content in organs of common buckwheat seedling.The results for control and MJ-treated plants were significantly different (P<0.01): for cotyledons and hypocotyls (4-and 7-days treated); for roots (7-days treated) .For roots (4-days treated) the differences were not significant.Seedlings prepared within 4-days sprouting in the darkness were transferred to the growth chamber and then treated with atmospheric MJ for 4-or 7-days.Chemical analyzes have been performed immediately after germination, and after 4 and 7 days of treatment.
Tested organ AAT level (fold change in relation to control) Cotyledons 3.60±0.11c Hypocotyl 5.22±0.34b Primary root 7.50±0.57a Phenylethylamine and metabolites in common buckwheat