SbCOMT (Bmr12) is involved in the biosynthesis of tricin-lignin in sorghum

Lignin in plant biomass represents a target for engineering strategies towards the development of a sustainable bioeconomy. In addition to the conventional lignin monomers, namely p-coumaryl, coniferyl and sinapyl alcohols, tricin has been shown to be part of the native lignin polymer in certain monocot species. Because tricin is considered to initiate the polymerization of lignin chains, elucidating its biosynthesis and mechanism of export to the cell wall constitute novel challenges for the engineering of bioenergy crops. Late steps of tricin biosynthesis require two methylation reactions involving the pathway intermediate selgin. It has recently been demonstrated in rice and maize that caffeate O-methyltransferase (COMT) involved in the synthesis syringyl (S) lignin units derived from sinapyl alcohol also participates in the synthesis of tricin in planta. In this work, we validate in sorghum (Sorghum bicolor L.) that the O-methyltransferase responsible for the production of S lignin units (SbCOMT / Bmr12) is also involved in the synthesis of lignin-linked tricin. In particular, we show that biomass from the sorghum bmr12 mutant contains lower level of tricin incorporated into lignin, and that SbCOMT can methylate the tricin precursors luteolin and selgin. Our genetic and biochemical data point toward a general mechanism whereby COMT is involved in the synthesis of both tricin and S lignin units.


Introduction
Lignin is a rigid and hydrophobic cell-wall polymer that played a central role in the evolutionary conquest of land by vascular plants. Lignin in angiosperms arises from the oxidative polymerization of phenylpropanoid-derived p-coumaryl, coniferyl and sinapyl alcohols, which leads to the formation of H, G, and S lignin units, respectively [1]. During the biosynthesis of these lignin monomers (or monolignols), the formation of sinapyl alcohol requires the 5-Omethylation of 5-hydroxyconiferaldehyde catalyzed by caffeate O-methyltransferase (COMT, EC 2.1.1.68) (Fig 1) [2,3].
Besides the presence of canonical H, G and S units, lignin exhibits compositional plasticity, as exemplified by the occurrence of the flavone tricin found in the lignin of several monocot species and the dicot alfalfa (Medicago sativa) [4]. In particular, tricin has been shown to react with monolignols under radical coupling conditions and the corresponding tricin-oligolignol metabolites were identified in maize extracts [5,6]. As a result, tricin monomers are found even in the highest molecular weight fractions of lignin and act as nucleation sites for lignification [5,6]. The biosynthesis of flavones starts with p-coumaroyl-CoA as a precursor, and tricin biosynthesis is achieved via 5'-O-methylation of selgin, which derives from chrysoeriol and luteolin (Fig 1) [7,8]. As examples, the two O-methyltransferases, OsCOMT1 from rice and ZmCOMT (encoded at the Bm3 locus) from maize, perform in vitro the 3'-O-methylation of luteolin to produce chrysoeriol [9][10][11]. Moreover, the affinity of OsCOMT1 and ZmCOMT toward selgin is also demonstrated by their capacity to form tricin via dual 3'/5'-O-methylation of tricetin (Fig 1) [8,11,12]. Consequently, rice seedlings of an OsCOMT1 T-DNA insertion mutant show reduction of methanol-extractable tricin [8], and biomass from the maize bm3 mutant has lower levels of lignin-linked tricin [13]. Finally, OsCOMT1 and ZmCOMT are also known to methylate 5-hydroxyconiferaldehyde and/or 5-hydroxyferulic acid, and transgenic rice and maize plants downregulated, respectively, for OsCOMT1 and ZmCOMT, exhibit lower amount of S lignin units [11,[13][14][15]. Overall, these observations suggest a general mechanism whereby COMT is involved in the synthesis of both tricin and S lignin units.  Tricin is produced and found in the lignin of sorghum [4], but none of the enzymes involved in the last steps of its biosynthesis have been characterized in this important multipurpose crop. The sorghum brown midrib12 (bmr12) mutant shows a reduction of S units in lignin due to a premature stop codon in the gene encoding for the COMT (SbCOMT) that methylates 5-hydroxyconiferaldehyde (Fig 1) [16][17][18]. Our objective in this work was to gain insight into the biosynthesis of tricin in sorghum and to determine the possible role of SbCOMT in the methylation step(s) of the tricin biosynthetic pathway.

Plant material
Biomass was harvested from field grown wild-type and bmr12 sorghum plants with the panicles removed as previously described [19]. Plants were grown in at the University of Nebraska Field Laboratory, Ithaca, NE (coordinates 41.163182, -96.410486). This land was rented to USDA-ARS sorghum project from the University of Nebraska Agriculture Research and Development Center (http://ardc.unl.edu). No special permission was required. The land has been in cropping systems for over 50 years. The endangered or protected species also do not apply [19].

Extraction of methanol-soluble metabolites
Ball-milled biomass from wild-type and bmr12 plants (50 mg) was mixed with 1 ml of 80% (v/ v) methanol-water and shaken at 1,400 rpm for 15 min at 70˚C. The mixture was cleared by centrifugation for 5 min, at 20,000 x g. This step was repeated five times. Extracts were pooled and cleared one more time by centrifugation (5 min, 20,000 × g), mixed with 3 mL of analytical grade water and filtered using Amicon Ultra centrifugal filters (3,000 Da MW cutoff regenerated cellulose membrane; EMD Millipore, Billerica, MA). An aliquot of the filtered extracts (1.5 mL) was dried under vacuum, re-suspended with 1 N HCl, and incubated at 95˚C for 3 h. The mixture was subjected to three ethyl acetate partitioning steps. Ethyl acetate fractions were pooled, dried in vacuo, and re-suspended in 50% (v/v) methanol-water (150 μL) prior to highperformance liquid chromatography (HPLC), electrospray ionization (ESI), and time-of-flight (TOF) mass spectrometry (MS) analysis.

Heterologous expression, purification and activity of SbCOMT
The pDONR221-SbCOMT entry vector was LR recombined with the pDEST17 bacterial expression vector, which introduces an N-terminal 6× His tag (Life Technologies, Foster City, CA). All vectors can be found through the Inventory of Composable Elements (ICE) at https:// acs-registry.jbei.org/. Rosetta 2 (DE3) E. coli (EMD Milipore, Billerica, MA) was used for protein expression. A single bacterial colony, grown on Luria-Bertani agar containing 100 µg/mL carbenicillin and 30 µg/mL chloramphenicol was used to inoculate a 5-mL liquid culture supplemented with the same antibiotic concentrations and grown overnight at 37˚C. The overnight culture was used to inoculate a 0.5-L Luria-Bertani culture at an OD 600 = 0.05 containing the same antibiotic concentrations and grown at 37˚C until it reaches an OD 600 = 0.8-1.0. Expression was induced by the addition of 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG), and the culture was transferred at 20˚C and grown for 24 h. The recombinant protein was affinity purified using a HIS-Select HF Nickel Affinity Gel (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions and buffer-exchanged with 50 mM Tris buffer pH 7.5 using Amicon Ultra centrifugal filters (10,000 Da MW cutoff regenerated cellulose membrane; EMD Millipore, Billerica, MA). Purity and integrity were verified by SDS-PAGE, and the recombinant protein was stored at −80˚C in 50 mM Tris buffer pH 7.5, containing 10% (v/v) glycerol.

Selgin synthesis
Selgin was synthesized as previously described [27]. Purity and integrity of the compound was validated by NMR and HPLC-ESI-TOF MS analyses ( Figure A in S1 File). The NMR spectrum was recorded on a Bruker AV-600.

Thioacidolysis
The release of tricin from cellulolytic lignin (5 mg) was conducted using the thioacidolysis procedure described in [4].

Results and discussion
Bmr12 sorghum biomass has reduced methanol-extractable tricin Methanol-soluble metabolites were extracted from total biomass of wild-type and bmr12 plants for the quantification of tricin and its biosynthetic precursors. Tricin and chrysoeriol amounts are reduced by more than 60% in the bmr12 mutant compared to wild-type plants, whereas luteolin and selgin contents are increased by 20% and 22%, respectively (Fig 2). Tricetin was not detected in wild-type and bmr12 plant extracts. These results suggest a role for SbCOMT in the biosynthesis of chrysoeriol and tricin, possibly via the methylation of luteolin. Values in bmr12 are expressed as a percentage of the values measured in wild-type extracts which correspond to 317 ± 4 µg/g dry weight (DW) for luteolin, 7.8 ± 0.0 µg/g DW for chrysoeriol, 2.0 ± 0.2 µg/g DW for selgin, and 274 ± 3 µg/g DW for tricin. Error bars represent the standard deviation from five experimental replicates. Asterisks indicate significant differences from the wild-type using the unpaired Student's t-test (*P < 0.05). https://doi.org/10.1371/journal.pone.0178160.g002 Bmr12 sorghum biomass has lower levels of lignin-linked tricin Cellulolytic lignin isolated from wild-type and bmr12 sorghum plant material was analyzed by 2D 13 C-1 H heteronuclear single quantum coherence (HSQC) NMR spectroscopy to determine the relative abundance of G, S, and tricin units incorporated in lignin (Fig 3). We observed in the lignin of the bmr12 plants a 50% reduction of S units and the presence of 5-hydroxyguaiacyl (5OH-G) units resulting from the incorporation of 5-hydroxyconiferyl alcohol. In addition, benzodioxane structures, which are typically formed during β-O-4 coupling of a monolignol with a 5OH-G unit, were detected only in the case of bmr12 ( Figure B in S1 File). In accordance with previously published data, these observations are consistent with a reduction of SbCOMT activity, which not only impacts the synthesis of sinapaldehyde and S lignin units, but also results in the accumulation of 5-hydroxyconiferaldehyde and 5OH-G lignin units [18]. Moreover, we report here that the relative amount of tricin in the lignin of bmr12 plants (~2%) is lower than that found in the lignin of wild-type plants (~5%) (Fig 3).
To support this observation, we quantified the absolute amount of tricin incorporated in the lignin of wild-type and bmr12 using thioacidolysis. The results showed that the lignin of wild-type plants contained 9.4 mg/g of tricin, which is consistent with previously published values obtained with this method [4], whereas the lignin of bmr12 plants contained only 2.5 mg/g of tricin (Fig 4). These data imply that, in addition to its role in the synthesis of S-lignin units, SbCOMT is involved in the synthesis of tricin-lignin.

SbCOMT (Bmr12) methylates luteolin, selgin, and tricetin
Recombinant his-tagged SbCOMT was produced in E. coli and purified for biochemical characterization to assess its role in tricin biosynthesis (Figure C in S1 File). Using S-adenosylmethionine as a methyl donor, incubations of recombinant SbCOMT with luteolin or selgin (custom synthesis) resulted in the synthesis of chrysoeriol and tricin, respectively (Fig 5A and 5B), by comparison with standard compounds (Fig 5C and 5D). None of these products was observed when the reactions were carried out with a pre-boiled enzyme preparation. These results indicate that SbCOMT is able to 3'-O-methylate luteolin and 5'-O-methylates selgin. The capacity of SbCOMT to perform 3-O-methylation has been previously reported using caffeic acid as a substrate [17]. Finally, we observed that incubation of SbCOMT with tricetin results in the synthesis of tricin (Fig 5E), which indicates that SbCOMT 5'-and 3'-O-methylates this substrate.

Conclusion
To conclude, we demonstrated in sorghum that the O-methyltransferase SbCOMT (Bmr12) involved in the synthesis of S lignin units also participates in the biosynthesis of the flavone tricin. Based on these results, chrysoeriol is a probable route for tricin synthesis in sorghum and the chrysoeriol 5'-hydroxylase involved in this route remains to be identified. Although our data cannot exclude the existence of a route via tricetin, to the best of our knowledge, tricetin has never been detected in sorghum. Lignin polymers incorporate tricin in several monocot species, including sorghum, and the sorghum bmr12 mutant exhibits lower levels of ligninlinked tricin in addition to a significant reduction of S units. These observations raise questions as to whether the contribution of COMT in the synthesis of both lignin monomers, tricin and sinapyl alcohol, is specific to certain species such as sorghum and maize or whether it represents Tricin was released from lignin using the thioacidolysis procedure and subsequently quantified by HPLC-ESI-TOF MS. Error bars represent the standard deviation from three experimental replicates. Asterisks indicate a significant difference from the wild-type using the unpaired Student's t-test (*P < 0.05).