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Graphical abstract Epichloënin A (1) has been isolated from fungal cultures together with a minor variant epichloënin B, and ferriepichloënin A and a related peptide epichloëamide have been found in endophyte-infected plants. Highlights ► Epichloënin A has been characterized as a ferrichrome octapeptide by HRMSn and NMR. ► The minor heptapeptide variant epichloënin B has been characterized by HRMSn. ► Ferriepichloënin A was detected in guttation fluid of endophyte-infected plants. ► The related peptide epichloëamide was also detected in guttation fluid. ► Epichloëamide has been characterized as an octapeptide by HRMSn., A number of genes encoding non-ribosomal peptide synthetases (NRPSs) have been identified in fungi of Epichloë/Neotyphodium species, endophytes of Pooid grasses, including sidN, putatively encoding a ferrichrome siderophore-synthesizing NRPS. Targeted gene replacement and complementation of sidN in Epichloë festucae has established that extracellular siderophore epichloënin A is the major product of the SidN enzyme complex (Johnson et al., 2007a). We report here high resolution mass spectrometric fragmentation experiments and NMR analysis of an isolated fraction establishing that epichloënin A is a siderophore of the ferrichrome family, comprising a cyclic sequence of four glycines, a glutamine and three Nδ-trans-anhydromevalonyl–Nδ-hydroxyornithine (AMHO) moieties. Epichloënin A is unusual among ferrichrome siderophores in comprising an octapeptide rather than hexapeptide sequence, and in incorporating a glutamine residue. During this investigation we have established that desferrichrome siderophores with pendant trans-AMHO groups can be distinguished from those with pendant cis-AMHO groups by the characteristic neutral loss of an hydroxyornithine moiety in the MS/MS spectrum. A minor component, epichloënin B, has been characterized as the triglycine variant by mass spectrometry. A peptide characterized by mass spectrometry as the putative deoxygenation product, epichloëamide has been detected together with ferriepichloënin A in guttation fluid from ryegrass (Lolium perenne) plants infected with wild-type E. festucae, but not in plants infected with the ΔsidN mutant strain, and also detected at trace levels in wild-type E. festucae fungal culture.

Based on direct infusion mass spectrometry we identified a novel alkaloid as a major component of perennial ryegrass (Lolium perenne). Initial mass spectral data suggested it to be a pyrrolizidine conjugate. As this class of alkaloids has not been described before from grasses, we isolated it to elucidate its structure. The isolated alkaloid proved to be a mixture of two stereoisomers. The structures of the two compounds as determined by 1D and 2D NMR spectroscopy, were E-thesinine-O-4′-α-rhamnoside (1) and Z-thesinine-O-4′-α-rhamnoside (2). These identifications were supported by the characterisation by GC-MS and optical rotation of (+)-isoretronecanol as the necine base released on alkaline hydrolysis of these alkaloids. 1 and 2 together with the aglycone and a hexoside were also detected in tall fescue (Festuca arundinacea). This is the first report of pyrrolizidine alkaloids produced by grasses (Poaceae).

A stereospecific glucosyltransferase, catalysing the transfer of glucose from UDP-glucose to the 4′-hydroxy-group of cis - p -coumaric acid, has been isolated and partially purified from Sphagnum fallax Klinggr. (Klinggr. clone 1) by ion exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The enzyme was purified 600-fold with a yield of 0.4%. The M r was estimated by gel filtration on a Superdex 200 column to be about 56 000. Optimal enzyme activity was observed in glycine-KOH at pH 9.3. The transferase activity was not influenced by divalent cations (1 mM) or EDTA (5 mM), but it was inhibited by NaCl (0.4 M) to 40% and by 2-mercaptoethanol (14 mM) to 30%. The enzyme showed a pronounced substrate specificity towards cis - p -coumaric acid with a K m value of 44 μM, trans - p -coumaric acid was not transformed. The apparent K m value for the cosubstrate UDP-glucose was 110 μM. The only reaction product was 4′-O-β- d -glucosyl -cis-p- coumaric acid. The substrate cis - p -coumaric acid inhibited the reaction at a concentration higher than 1 mM. The only other phenolic substrate transformed by the purified glucosyltransferase was cis -caffeic acid, with an enzyme activity of 10% compared with that using cis - p -coumaric acid as substrate.

Exogenously applied p -coumaric acid (0.1 mM) was rapidly taken up by Sphagnum fallax batch cultures, precultivated axenically in bioreactors, and metabolized to trans -sphagnum acid and a p -coumaric acid conjugate. The structure of the isolated conjugate was elucidated by NMR and IR spectroscopy and was shown to be 4′-O-β- d -glucosyl -cis-p- coumaric acid. Small amounts (80–160 nmol g −1 dry wt) of this component are also always detectable in the homogenate of untreated S. fallax plantlets. Acid hydrolysis (N HCl) of the isolated glucoside or treatment with a β-glucosidase from almonds yielded mainly cis - p -coumaric acid. Irradiation of the cis -glucoside with UV light (λ = 254 nm) resulted in the formation of 4′-O-β- d -glucosyl -trans-p- coumaric acid. The biosynthesis and accumulation of 4′-O-β- d -glucosyl -cis-p- coumaric acid is dependent on the availability of cis - p -coumaric acid in the external medium; exogenously in the dark applied trans - p -coumaric acid is not glucosylated, whereas trans -sphagnum acid is synthesized from trans - p -coumaric acid and accumulated in the light as in the dark. A cytosolic uridine diphosphate glucose (UDPG): p -coumarate glucosyltransferase, which exhibits an unusual substrate specificity for cis - p -coumaric acid, was isolated from Sphagnum fallax . The biosynthesis of phenylpropanoids in Sphagnum known up to now is discussed based on the present results.

[ E ]-4-ethoxycarbonyl-3-(4′-hydroxyphenyl)-but-2-en-1-carboxylic acid ( trans -sphagnum acid ethyl ester) and p -hydroxyacetophenone were isolated from ethanolic extracts of Sphagnum magellanicum cell walls and [ Z ]-3-(4′-hydroxyphenyl)-pent-2-en-1,5-dicarboxylic acid ( cis -sphagnum acid) from irradiated trans -sphagnum acid solutions. The structures of these compounds were elucidated by means of NMR spectroscopy. Compartmentalization studies revealed that the main part of detectable sphagnum acid is buffer-soluble, as well as in Sphagnum collected from natural sites and in Sphagnum cultivated in bioreactors. Cultivating Sphagnum species in bioreactors under axenic conditions leads to an enhancement of the buffer-soluble part of all analysed phenolics with a concomitant decrease of their cell wall-bound amount. Sphagnum acid, sphagnum acid ethyl ester, hydroxybutenolide, p -hydroxybenzoic acid, p -coumaric acid and t -cinnamic acid are also excreted to a remarkable extent into the effluent culture media of S. fallax and S. cuspidatum . Specific compartments for the storage of endogenous free water-soluble phenolics are postulated.