Arabidopsis thaliana

Strehmel, Nadine, Böttcher, Christoph, Schmidt, Stephan & Scheel, Dierk, 2014, Profiling of secondary metabolites in root exudates of Arabidopsis thaliana, Phytochemistry 108, pp. 35-46 : 38-43

2.4. Compounds identified in root exudates of A. thaliana

2.4.1. Nucleosides, amino acids and dipeptides

Although the established method was not intended to target polar metabolites, several polar aromatic compounds including three nucleosides (1–3), two deoxynucleosides (4, 5), a nucleoside derivative (6) and the aromatic amino acids Phe (7), Tyr (8) and Trp (9) were detected in root exudates of A. thaliana . In addition, a simple β- carboline alkaloid (10) previously reported to accumulate in tomato fruits ( Yahara et al., 2004) was identified using a synthetic reference compound prepared from Trp and formaldehyde ( Tilstra et al., 1990). While other proteinogenic amino acids were not detected using the established analytical method, the more hydrophobic side chain-elongated Met homologs (11, 12) and their S - oxides (13, 14) were putatively identified based on the elemental composition, the presence of three exchangeable protons and the occurrence of characteristic neutral losses of 46.01 Da (HCOOH) and 48.00 Da ( CH 3 SH)/64.00 Da ( CH 3 SOH) in the CID mass spectra of the [M+H] + ions. Compounds 11–14 represent biosynthetic intermediates of corresponding ω- methylsulfinylalkyl/ω- methylthioalkyl glucosinolates of chain length 7 and 8 which are known constituents in roots of the accession Col-0 ( Brown et al., 2003; Petersen et al., 2002).

Beside nucleosides and amino acids, an array of 20 dipeptides (15–34) composed of the amino acids Gly, Val, Leu, Ile, Pro, Phe and Tyr was identified in root exudates. Structural assignments are in agreement with the elemental composition, the number of exchangeable protons and the occurrence of characteristic y-type fragment ions upon CID of the [M+H] + ions. To discriminate Leufrom Ile-containing peptides, a total of 16 dipeptides were synthesized and used for authentication. The spectrum of dipeptides detected in root exudates matches the set of 14 dipeptides reported to accumulate in roots of A. thaliana in a cell-type specific manner ( Moussaieff et al., 2013).

2.4.2. Degradation products of methionine-derived glucosinolates

Met-derived glucosinolates are the predominating phytoanticipins in most organs of A. thaliana . Despite their significant accumulation level in root tissue ( Brown et al., 2003; Petersen et al., 2002) glucosinolates were not consistently detected in root exudates using the established experimental setup, which is in contrast to a previous study on root exudates of Brassica rapa ( Schreiner et al., 2011) . Instead of glucosinolates, a set of 8 putative glucosinolate degradation products (35–42) derived from 8-methylthiooctyl, 8-methylsulfinyloctyl and 7-methylsulfinylheptyl gluco sinolate were identified based on the occurrence of characteristic neutral losses of 48.00 Da and 64.00 Da upon CID of the [M+H] + ions which are indicative for methylthio and methylsulfinyl moieties, respectively. Among them, classical glucosinolate breakdown products known to be produced upon tissue damage were found including two nitriles (35, 36) and an isothiocyanate (37). Related to these compounds, a carboxylic acid (38) and its amide (39) as well as three amines (40–42) were identified. It was suggested, that these compounds represent glucosinolate breakdown products, which are formed in intact tissue by either nitrilase-mediated hydrolysis of nitriles or by degradation of isothiocyanate–glutathione conjugates ( Bednarek et al., 2009; Wittstock and Burow, 2010).

2.4.3. Degradation products of tryptophan-derived glucosinolates and other indolic compounds

In addition to aliphatic glucosinolates indolic glucosinolates accumulate in A. thaliana and, in particular in roots, relatively large amounts of these compounds are found ( Brown et al., 2003). As observed for Met-derived glucosinolates, the major Trp-derived glucosinolates of roots (indol-3-yl methyl, 4-methoxy and 1-methoxyindol-3-yl methyl glucosinolate) were absent in root exudates. Again, corresponding degradation products including indol-3- ylmethyl amine (43), its 4-methoxy (44) and 1-methoxy derivative (45) were detected. Here, isomers 44 and 45 can be discriminated based on the number of exchangeable protons determined for the dominating [M+H-NH 3] + - type in-source fragment ion. Amine 43 was recently identified as a product of the atypical myrosinase PENETRATION2 (At 2g 44490) which is involved in an inducible pre-invasion resistance mechanism conferring broad-spectrum resistance to fungal pathogens ( Bednarek et al., 2009; Lipka et al., 2005). In addition, further indolic compounds were identified in root exudates including indole-3-carbaldehyde (46), 4-hydroxyindole-3-carbaldehyde (47), indole-3-carboxylic acid (48), a malonyl-glucoside of 6-hydroxyindole-3-carboxylic acid (49) and an unknown indole derivative (50). Compounds 46–49 represent known metabolites which constitutively accumulate (in case of 47 and 48 as glucose conjugates) in leaf and root tissue of A. thaliana and exhibit a pathogen-inducible accumulation pattern ( Bednarek et al., 2005; Bottcher et al., 2014; Hagemeier et al., 2001).

It should be noted that camalexin, the major indolic phytoalexin of A. thaliana , was not detected as root exudate in sterile nutrient solutions. In accordance to literature data describing the exudation of this phytoalexin following treatment with a conserved 22- amino-acid peptide from bacterial flagellin (flg22) ( Millet et al., 2010), camalexin became clearly detectable when analyzing microbially contaminated nutrient solutions. This suggests that the camalexin level could be used as an additional marker to judge the sterility of the hydroponic culture system.

2.4.4. Salicylic and jasmonic acid degradation products

Based on their elemental composition and the occurrence of consecutive neutral losses of 132.04 Da (C 5 H 8 O 4) and 43.99 Da (CO 2) upon CID of the [M – H] – ions, two exuded compounds (51, 52) were annotated as dihydroxybenzoic acid (DHBA) pentosides. Pentoside 51 was identified as 2,3-DHBA 3- O -β- D- xyloside using an authenticated standard ( Bartsch et al., 2010) whereas the other was annotated as 2,5-DHBA conjugate based on acid-catalyzed hydrolysis experiments. As recently shown, both DHBA pentosides represent the major salicylic acid catabolites in young and senescent A. thaliana rosette leaves ( Zhang et al., 2013). In addition, another plant hormone catabolite (53) was detected in root exudates (Supplementary Fig. 5A). The CID mass spectrum obtained from the [M – H] – ion of 53 exhibited a prominent neutral loss of 79.96 Da (SO 3) and a corresponding fragment ion at m / z 96.96 (HSO 4 –) indicating the presence of a sulfate moiety (Supplementary Fig. 5B). The elemental composition of 53 was determined to be C 12 H 20 O 7 S suggesting that this compound might represent a sulfated dihydro-hydroxyjasmonic acid derivative. To prove this hypothesis, A. thaliana seedlings were grown in liquid culture, treated for 24 h with [2-D 2]-jasmonic acid, extracted and analyzed by UPLC/ESI-QTOFMS. Following this treatment, 53 and its deuterated isotopolgue became detectable (Supplementary Fig. 5C). On-line H/ D exchange experiments revealed the presence of two exchangeable protons (Supplementary Fig. 5D). Thus, compound 53 possesses a saturated side chain and a ring keto group rather than an unsaturated side chain and a ring hydroxy group. Beside 12- hydroxyjasmonic acid O -sulfate ( Gidda et al., 2003), compound 53 represents a novel sulfated jasmonic acid catabolite produced in A. thaliana .

2.4.5. Phenylpropanoids

2.4.5.1. Lignols. Coniferin (54) and syringin (55) represent the predominant phenylpropanoids in dark-grown roots of A. thaliana ( Bednarek et al., 2005) and were also detected in root exudates. Both monolignol glucosides were accompanied by related di- and trilignols and derivatives thereof. The identity and the linkage type of these compounds was derived from the analysis of CID mass spectra obtained from corresponding [M – H] – ions ( Morreel et al., 2010a,b). The CID mass spectra of the β- aryl ether-type dimers 56 and 57 carrying an 8- O -4 linkage exhibited a characteristic neutral loss of 48.02 Da according to a combined loss of water and formaldehyde from the [M – H] – ion. In addition, formation of A – and B – fragment ions by cleavage of the aryl ether bond facilitated the identification of the aromatic units of these dimers ( Morreel et al., 2010b). For the phenylcoumaran-type dimers 58 and 59 with 8–5 linkage, fragmentation of the [M – H] – ions resulted in dominating neutral losses of 18.01 Da (H 2 O) and 30.01 Da ( CH 2 O) ( Morreel et al., 2010b). Three resinol-type dilignols with 8–8 linkage including syringaresinol (60), lariciresinol (61) and a derived hexoside (62) were found in root exudates. In accordance with the literature, the CID mass spectrum obtained from deprotonated 60 showed the product ions [M – H-CH 3]. – and [M – H-CH 2 O] – as well as a 2,5 X – fragment ion at m / z 181.05 ( Morreel et al., 2010b). The accumulation of glycosylated syringaresinol and lariciresinol in roots of A. thaliana was described ( Matsuda et al., 2010; Nakatsubo et al., 2008). Based on the fragmentation characteristics observed for dilignols, three trilignols (63–65) with coniferyl and syringyl alcohol as monomeric untis were tentatively identified based on the fragmentation characteristics observed for corresponding dilignols.

2.4.5.2. Coumarins. The coumarin glucoside scopolin represents another abundant root phenylpropanoid ( Bednarek et al., 2005; Kai et al., 2006). It was recently shown, that the biosynthesis of coumarins in roots and their exudation can be stimulated inter alia upon iron deprivation indicating that coumarins are involved in the acquisition of iron in A. thaliana ( Fourcroy et al., 2014; Schmid et al., 2014; Schmidt et al., 2014).

Esculetin (66), scopoletin (67) and its glucosides esculin (68) and scopolin (69) were detected in root exudates and identified by authenticated standards. Beside these coumarins which are already known to be exuded from roots ( Fourcroy et al., 2014; Schmid et al., 2014), further esculetin and scopoletin conjugates were tentatively identified in root exudates on the basis of their mass spectral fragmentation pattern. Scopoletin was found to be conjugated to a hexose–pentose and to a benzoylated hexose–pentose as indicated by a neutral loss of 294.10 Da (C 11 H 18 O 9) from the [M – H] – ion of 70 and consecutive neutral losses of 122.04 Da (C 7 H 6 O 2) and 294.10 Da from the [M – H] – ion of 71, respectively. In addition, an 8- O -4-linked coniferylalcohol–scopoletin conjugate (72) was annotated based on the occurrence of a characteristic neutral loss of 48.02 Da from its [M – H] – ion and characteristic A – and B – fragment ions at m / z 195.07 and m / z 191.03. Compounds 73 and 74 were tentatively identified as 8- O -4-linked cross-coupling products of coniferyl alcohol with esculetin and syringyl alcohol with esculetin, respectively. The presence of an unsubstitued 3- OH group in the esculetin moiety thereby results in the formation of a benzodioxane-type linkage motif. In accordance with the literature ( Morreel et al., 2010b), CID of deprotonated 73 and 74 resulted in neutral losses of water and formaldehyde from the [M – H] – ion and formation of A – and B – fragment ions at m / z 179.07 and m / z 177.02 for 73 and m / z 209.08 and m / z 177.02 for 74. In addition, a scopoletin dehydrodimer (75) with unknown linkage motif and an unknown oligolignol (76) with four phenylpropanoid units of which one was identified as scopoletin were detected in root exudates.

2.4.5.3. Hydroxycinnamic acid conjugates and flavonoids. Although phenolic acids represent well-known constituents of root exudates ( Lanoue et al., 2010), free hydroxycinnamic and hydroxybenzoic acids could not be detected in root exudates of A. thaliana using the established experimental system. Due to the low process efficiencies found for cinnamic and salicylic acid (vide supra), it remains unclear, whether A. thalina roots do not exude these compounds under sterile conditions or their detection is hampered by the sample preparation protocol. However, three monolignol-ferulic acid conjugates (77, 78, 79) and a ferulic acid dimer (80) were identified. Interestingly, two of them (77, 79) were detected as sulfate conjugates as indicated by a neutral loss of 79.96 Da from the [M – H] – ions. For the 8- O -4 cross-coupled coniferyl alcohol–ferulic acid conjugate 77 a characteristic neutral loss of 48.02 Da from the [M – H-SO 3] – ion was registered together with A – and B – fragments at m / z 195.06 and m / z 193.05. As observed for phenylcoumaran-type dilignols, the CID mass spectra of the 8–5 cross-coupled coniferyl alcohol–ferulic acid conjugates 78 and 79 are characterized by neutral losses of water and formaldehyde from the [M – H] – or [M – H-SO 3] – ion, respectively.

Another sulfate conjugate was compound 81. Calculation of the elemental composition of 81 suggested the presence of three nitrogen atoms. The CID mass spectrum obtained from [M+H] + of 81 revealed neutral losses of 17.03 Da (NH 3), 74.08 Da (C 3 H 10 N 2), 88.10 Da (C 4 H 12 N 2) and 145.16 Da (C 7 H 19 N 3) from the [M+H-SO 3] + ion, the latter resulting in a fragment ion at m / z 291.07 with the elemental composition of C 18 H 11 O 4 +. This suggests that 81 represents a sulfated cyclic didehydro-di(coumaroyl)spermidine conjugate. The Arabidopsis genome contains a gene (At 2g 25150) encoding a functional coumaroyl-CoA:spermidine coumaroyltransferase ( Luo et al., 2009). This gene was shown to exhibit a root specific expression pattern and its overexpression resulted in the accumulation of di(coumaroyl)spermidine and a related hexoside in roots ( Luo et al., 2009). Compound 81 could represent a metabolite which might be synthesized from di(coumaroyl)spermidine by intramolecular oxidative phenol coupling and subsequent sulfation.

Although flavonol glycosides are absent in dark-grown roots in A. thaliana ( Bednarek et al., 2005; Hemm et al., 2004), a kaempferol triglycoside (82), which was found to be accumulating in rosette leaves ( Yonekura-Sakakibara et al., 2008) was identified in root exudates.

2.4.6. Fatty acid derivatives

Among the more hydrophobic compounds of the semipolar root exudate fraction three oxygenated C18 fatty acids (83–85) were detected. For compounds 83 and 84 on-line H/D exchange experiments revealed the presence of four exchangeable protons indicating that both fatty acid derivatives are functionalized by three hydroxyl groups. The CID mass spectra obtained from the [M – H] – ion of 83 and 84 showed fragment ions at m / z 229.14 (C 12 H 21 O 4 –) and m / z 171.10 (C 9 H 15 O 3 –) suggesting that both fatty acid derivatives are hydroxylated in position 12 and/or 13 and 9 and/or 10. In a recent study, 9,12,13-trihydroxy-10,15-octadecadienoic acid and 9,12,13-trihydroxy-10-dodecenoic acid were identified as infection markers in the leaf apoplast washing fluid of A. thaliana following infection by Verticillium longisporum ( Floerl et al., 2012) . Authenticated standards of both compounds exhibited identical chromatographic and mass spectral properties as observed for 83 and 84.

The elemental composition of the fatty acid derivative 85 indicated four oxygen atoms and four double bond equivalents, while on-line H/D exchange experiments revealed the presence of only two exchangeable protons. Unfortunately, the CID mass spectrum obtained from deprotonated 85 showed only unspecific neutral losses of water and carbon dioxide for which reason 85 could not be characterized further.

In contrast, the CID mass spectrum obtained from [M+H] + of 86 exhibited characteristic fragments at m / z 184.07 (C 5 H 15 NO 4 P +) and m / z 104.11 (C 5 H 15 NO + 4) indicating the presence of a phosphorylcholine moiety. The occurrence of fragments ions at m / z 187.10 (C 9 H 15 O 4 –), m / z 169.09 (C 9 H 13 O 3 –) and m / z 125.10 (C 8 H 13 O –) in the CID mass spectrum obtained from deprotonated 86 was in accordance with a lysophosphatidylcholine of an oxygenated C9 fatty acid. Since on-line H/D-exchange experiments revealed the presence of two exchangeable protons 86 is postulated to be an azelaoyl lysophosphatidylcholine. Azelaic acid itself was shown to be produced by oxidative fragmentation of esterified 18:2 and 18:3 fatty acid species and proposed to be a general marker for lipid peroxidation ( Zoeller et al., 2012). In addition, this dicarboxylic acid was identified as pathogen inducible metabolite from the vascular sap of A. thaliana and shown to confer local and systemic resistance against the pathogen Pseudomonas syringae ( Jung et al., 2009) .

2.4.7. Unknown components

Among the compounds which were reproducibly detected in root exudates of A. thaliana , 17 could not or only partially be structurally characterized. Thereof, 11 compounds were annotated as glycoconjugates with unknown aglycone based on characteristic neutral losses and fragment ions. Among them, five hexose conjugates (87, 91, 92, 93, 95), three malonyl–hexose conjugates (88, 94, 97), a hexose–deoxyhexose conjugate (89) and a malonyl–hexose– deoxyhexose conjugate (90) were detected. Beside the three sulfated phenylpropanoids and the sulfated jasmonic acid derivative, another sulfate conjugate (98) was found.