Methylenetetrahydrofolate reductase modulates methyl metabolism and lignin monomer methylation in maize

Methylenetetrahydrofolate reductase modulates methyl metabolism and lignin monomer methylation in... Abstract The brown midrib2 (bm2) mutant of maize, which has a modified lignin composition, contains a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. Here, we show that a MITE transposon insertion caused down-regulation of MTHFR, with an accompanying decrease in 5-methyl-tetrahydrofolate and an increase in 5, 10-methylene-tetrahydrofolate and tetrahydrofolate in the bm2 mutant. Furthermore, MTHFR mutation did not change the content of S-adenosyl methionine (SAM), the methyl group donor involved in the biosynthesis of guaiacyl and syringyl lignins, but increased the level of S-adenosyl homocysteine (SAH), the demethylation product of SAM. Moreover, competitive inhibition of the maize caffeoyl CoA O-methyltransferase (CCoAOMT) and caffeic acid O-methyltransferase (COMT) enzyme activities by SAH was found, suggesting that the SAH/SAM ratio, rather than the concentration of SAM, regulates the transmethylation reactions of lignin intermediates. Phenolic profiling revealed that caffeoyl alcohol glucose derivatives accumulated in the bm2 mutant, indicating impaired 3-O-methylation of monolignols. A remarkable increase in the unusual catechyl lignin in the mutant demonstrates that MTHFR down-regulation mainly affects guaiacyl lignin biosynthesis, consistent with the observation that CCoAOMT is more sensitive to SAH inhibition than COMT. This study uncovered a novel regulatory mechanism in lignin biosynthesis, which may offer an effective approach to utilizing lignocellulosic feedstocks in the future. Lignin, maize, methylenetetrahydrofolate reductase, methyl metabolism, S-adenosyl homocysteine, S-adenosyl methionine Introduction Maize (Zea mays L.) originated in Central America and is one of the most widely grown crops in the world. Corn stover is a lignocellulosic feedstock that can be utilized to produce renewable energy. Brown midrib (bm) mutants of maize are spontaneous mutants that have reddish-brown vascular tissues in the leaf midrib and stalk. Among them, the bm3 mutant has been used in commercial hybrids because of its high cell wall digestibility (Sattler et al., 2010). Six bm mutants (bm1–6) have been identified in maize since the 1930s (Ali et al., 2010). To date, the bm1, 2, 3, and 4 loci have been identified to encode genes that can affect lignin biosynthesis (Vignols et al., 1995; Chen et al., 2012b; Tang et al., 2014; Li et al., 2015). Lignin is a complex phenolic polymer that contributes to structural support, water transport, and biotic and abiotic stress defense during plant growth and development (Boerjan et al., 2003). Examination of lignin-altered maize, switchgrass, Brachypodium, Arabidopsis, alfalfa, and poplar has shown that lignin modification can substantially improve forage digestibility, bioethanol production, and pulping efficiency (Pilate et al., 2002; Barrière et al., 2006; Chen and Dixon, 2007; Fu et al., 2011a, 2011b; Ho-Yue-Kuang et al., 2016; Smith et al., 2017). The monolignols, the lignin precursors, are synthesized through the hydroxylation and methoxylation of derivatives from the phenylpropanoid pathway. Three principal monolignols, named p-coumaryl, coniferyl, and sinapyl alcohols, are incorporated into lignin polymers to produce p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively (Boerjan et al., 2003). Their proportions vary among plant species and tissue types, but the abundance of G and S units is generally very high compared with the H unit (Chapple et al., 1992). Genetic manipulation of the ratio of G to S in the cell wall is complex and involves the regulation of both caffeoyl CoA O-methyltransferase (CCoAOMT) and caffeic acid O-methyltransferase (COMT) (Zhong et al., 1998; Li et al., 2000). The coordinated regulatory mechanism acting on the O-methyltransferases responsible for the biosynthesis of catechyl (C) and G lignins, however, still remains largely unknown. S-adenosyl methionine (SAM), as the primary methyl donor, is required for the biosynthesis of both G and S monolignols (Hanson et al., 2000), and therefore genetic regulation of SAM biosynthesis has the potential to reduce the extent of O-methylation of G and S monolignols (Shen et al., 2002). Previous studies in animals and microbes, however, have shown that S-adenosyl homocysteine (SAH), the demethylation product of SAM, can inhibit the enzymatic activities of methyltransferases by competing with SAM for the binding pocket (Keating et al., 1991; Nguyen et al., 2001). Thus, the ratio of SAH to SAM, rather than the level of SAM, is a crucial factor controlling the methylation status of these cells. However, the effects of SAM and SAH on methyltransferase activity have yet to be widely investigated in higher plants. The methyl moiety of SAM is derived from one-carbon (C1) metabolism and biosynthesized through folate-mediated reactions and the methionine (Met) cycle (Hanson et al., 2000; Hanson and Roje, 2001; Bradbury et al., 2014). C1 metabolism is an essential biological process in plants from green algae to angiosperms. The C1 units in different oxidation states (10-formyl-THF, 5-formyl-THF, 5,10-methenyl-THF, 5,10-methylene-THF, and 5-methyl-THF) are carried by tetrahydrofolate (THF). These C1 derivatives of THF are enzymatically interconverted between different oxidation states (Hanson and Roje, 2001). Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is required to convert 5,10-methylene-THF to 5-methyl-THF, which donates the methyl group to homocysteine (Hcy) for the synthesis of Met and then SAM (Roje et al., 1999). The vast majority of the flux of methyl groups through SAM in lignin-producing cells is used for the methylation of lignin monomers (Hanson and Roje, 2001). Although limited biochemical characterization of MTHFRs has previously been conducted with enzymes from Arabidopsis and maize, their detailed biological functions merit further investigation (Roje et al., 1999). Recently, an MTHFR-encoding gene (GRMZM2G347056, MTHFR-1) has been identified as the bm2 locus in maize (Tang et al., 2014). In the bm2 mutant, lignin accumulation is significantly reduced, and a brownish pigmentation first appears in the leaf midribs around the V4 stage (Tang et al., 2014). This mutation therefore points to the function of MTHFR in lignin biosynthesis. A predicted mechanism based on the possible effects of MTHFR on SAM biosynthesis is currently used to explain the impairment of lignin biosynthesis in the bm2 mutant (Tang et al., 2014), but in the absence of experimental evidence it remains largely unclear how MTHFR influences the flux of methyl groups towards the biosynthesis of monolignols and their derivatives. It has been shown, however, that regulation of MTHFR can affect the N-demethylation of nicotine markedly without significantly altering the contents of SAM and Met and without perturbing plant growth in transgenic tobacco plants in which MTHFR is down-regulated (Hung et al., 2013). To elucidate the function of MTHFR in methyl metabolism and lignin biosynthesis in maize, we cloned the full-length genomic sequence of MTHFR-1 from the bm2-ref mutant and recovered an insertion of a miniature inverted-repeat transposable element (MITE) from the 5ʹ untranslated region (5ʹUTR) that can cause a substantial decrease in MTHFR-1 transcript abundance. Our results showed that down-regulation of MTHFR-1 affected the accumulation of 5,10-methylene-THF and THF, 5-methyl-THF, SAH, and Hcy, but not Met and SAM, suggesting an important role of SAH in the regulation of transmethylation reactions of lignin intermediates. As a consequence, the O-methylation of lignin monomers was impaired, resulting in a significant reduction in G lignins and a remarkable accumulation of novel phenolics and unusual C lignins that derive from the polymerization of caffeyl alcohol in bm2 mutants; however, the content of S lignins was little changed. Furthermore, the altered lignin composition had no impact on total lignin content and plant growth, but led to a significant improvement in the efficiency of cell wall saccharification. Our findings suggest that MTHFR is a potential target for regulating the biosynthesis of G and C lignins, and may open up another avenue for lignin engineering of fuel and feed crops. Materials and methods Plant materials and growth conditions The maize stocks, 114A (bm2-PI586725), 134E (bm2-Mu-10-7061A), and 134K (bm2-Mu-10-7073G) containing the bm2 allele, were obtained from the Maize Genetics COOP Stock Center. The bm2-ref near-isogenic line, which was developed following six backcrosses of 114A with B73 as described by Vermerris et al. (2010), was used for functional characterization of MTHFR-1. The bm2-Mu mutant lines (134E and 134K) were used for soluble phenylpropanoid profiling analysis. Plant phenotype measurement was performed on maize plants grown in a greenhouse at 26 °C with 16 h light (390 µmol m–2 s–1)/8 h dark. Analysis of gene transcript abundance MTHFR-1 (GRMZM2G347056_V3, Zm00001d034602_V4) sequences were isolated from the bm2-ref mutant and the wild-type (WT) B73. Three primer pairs, designed in the 5ʹ untranslated region (UTR), open reading frame (ORF), and 3ʹUTR of MTHFR-1, were used to detect the expression level of MTHFR-1 in midribs by quantitative real-time reverse transcription–PCR (qRT–PCR) as described by Tang et al. (2014). The primers used for qRT–PCR are listed in Supplementary Table S1 at JXB online. Luciferase activity assay The 5ʹUTR and 1.5 kb promoter regions of MTHFR-1 were isolated from bm2-ref and B73. The 347 bp MITE insertion in the 5ʹUTR of MTHFR-1 was fused into the 5ʹUTR of MTHFR-1 from B73 at the same location as in the bm2-ref gene. The above three 5ʹ non-coding regions (NCRs) were cloned into the pGreenII 0800-LUC vector (Hellens et al., 2005), and the resulting constructs were transformed into maize leaf protoplasts as described by Sheen (1991). The relative ratio of firefly luciferase to Renilla luciferase was determined using a dual-luciferase reporter assay system (Promega). Determination of the intermediates in methyl metabolism Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants. The fresh samples were homogenized in liquid nitrogen and used for determination of the 5-methyl-THF and 5,10-methylene-THF contents by HPLC according to Hung et al. (2013). Immunoassay of Met, SAM, SAH, and Hcy was conducted as described by Hao et al. (2016). Microarray analysis and qRT–PCR validation The midribs were separated from the leaves of 60-day-old bm2-ref mutants and B73 WT plants. RNA extraction and purification, probe labeling, hybridization, and scanning for microarray analysis were conducted as previously described by Fu et al. (2012). The transcript abundance of differentially expressed probe sets related to C1 metabolism and the lignin pathway was validated by qRT–PCR. The primers used for qRT–PCR are listed in Supplementary Table S1. Assay of CCoAOMT and COMT activity Powdered fresh midrib tissues of B73 plants (~500 mg) were extracted for 3 h at 4 °C in extraction buffer (Liu et al., 2012). The samples were centrifuged at 17900 g for 20 min at 4 °C, and the extracts were desalted on PD-10 columns (Pharmacia). The ORF sequences of CCoAOMT2 (GRMZM2G099363_V3, Zm00001d045206_V4) and COMT (AC196475.3_FG004_V3, Zm00001d049541_V4) isolated from B73 were cloned into the pET28a vector to produce recombinant proteins in transformed Escherichia coli. Caffeoyl CoA and 5-OH coniferyl alcohol were used as substrates with crude enzyme extracts. The enzyme activity assay for CCoAOMT and COMT was performed as described by Liu et al. (2012). Biochemical characterization of the bm2 mutant Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants, homogenized in liquid nitrogen, and lyophilized. Polar and non-polar metabolite profiling of bm2-ref and B73 were performed by gas chromatography-mass spectrometry (GC-MS) as described by Broeckling et al. (2005). The methanolic extract from lyophilized materials including bm2-ref mutants, bm2-Mu mutants, and B73 WT plants was subjected to soluble phenolics profiling analysis by reversed-phase liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry (Fu et al., 2011b). The phenolic glucoside derivatives were degraded by β-glucosidase according to Tian and Dixon (2006) and its hydrolysis products were identified on the basis of their UV-visible spectra, mass spectra, and comparison with authentic standard compounds. Caffeyl alcohol and 5-OH coniferyl alcohol were synthesized by Chemistry Research Solution LLC (PA, USA), and p-coumaric acid was ordered from Sigma-Aldrich (St Louis, MO, USA). Lyophilized extractive free cell wall residues (CWRs) were used for lignin analysis. The acetyl bromide (AcBr) method (Hatfield et al., 1999) and the thioacidolysis method (Lapierre et al., 1995) were used to quantify the lignin content and composition of maize materials, respectively. Lignins were extracted from cell walls of bm2-ref and B73 plants as described by Funaoka and Fukatsu (1996), and were subjected to two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (2D HSQC NMR) analysis as described by Tobimatsu et al. (2013). Measurement of saccharification efficiency of cell walls Stalk samples were collected from bm2-ref mutants and B73 WT plants at the R1 stage (silk emergence) and dried in an oven at 40 °C for 1 week. Samples were ground through a Wiley mill with a 1 mm sieve, and the extractive free CWRs were used for saccharification efficiency analysis. Saccharification of the maize samples was performed following the analytical procedure described by Fu et al. (2011a). Briefly, solubilized sugars were yielded from CWRs digested by pretreatment with 1.5% H2SO4 at 121 °C for 40 min followed by washing with Milli-Q water and then exposure to a cellulase and cellobiase mixture for 72 h. The solubilized sugars were detected by the phenol–sulfuric acid assay method (Dubois et al., 1956). Statistical analysis Six biological replicates were used for analyzing the contents of Met, SAM, SAH, Hcy, and polar and non-polar metabolites in the bm2-ref mutant and B73 WT plants; for all other experiments, samples were collected from three biological replicates. The mean values were used for statistical analyses. Data from each trait were subjected to Student’s t-test. The significance of treatments was tested at the P=0.05 and 0.01 levels. Standard errors are provided in all tables and figures as appropriate. Results A MITE insertion reduced bm2 transcription efficiency Previous research has shown that the bm2 gene encodes a functional MTHFR (MTHFR-1) but its transcript abundance is substantially reduced in the mutant (Tang et al., 2014). To investigate the cause of the low MTHFR-1 transcript abundance, we generated the bm2-ref near-isogenic line in the B73 background after six backcrosses of 114A with B73. Our qRT–PCR analysis showed that the MTHFR-1 transcript abundance in the bm2-ref mutant was reduced to 1.4% of the control line value for the 5ʹ UTR and ~30% for the ORF and the 3ʹUTR (Fig. 1A). Furthermore, we isolated the MTHFR-1 genomic sequences from the bm2-ref mutant and WT (B73) maize. Two tandem duplications of the MITE insert were found in the first MTHFR-1 intron in the WT plants. A 414 bp deletion containing one of these duplications occurred in the same MTHFR-1 region in the bm2-ref mutant (Fig. 1B). Alignment between the full-length cDNA sequences of MTHFR-1 from the bm2-ref mutant and B73 revealed that the ORF sequence was the same except for a synonymous mutation (Fig. 1B). In addition to the deletion, a 347 bp insertion in the 5ʹUTR of MTHFR-1 was identified in the mutant (Fig. 1B–D, Supplementary Fig. S1). Bioinformatic analysis further revealed that the insertion sequence contains stem-loop structures and belongs to the Tourist-like MITE family, which may explain why 5ʹUTR transcripts of MTHFR-1 were barely detected in the bm2-ref mutant by qRT–PCR (Fig. 1E). Additionally, this type of MITE inserts only into introns and 3ʹUTRs, but not exons and 5ʹUTRs in the B73 maize genome (Supplementary Table S2). Fig. 1. View largeDownload slide A MITE insertion in the 5ʹUTR reduced bm2 transcripts dramatically. (A) Relative transcript abundance of MTHFR-1 in the bm2-ref mutant and B73 (wild-type). Primers designed on the basis of the 5ʹUTR, ORF, and 3ʹUTR sequences were used in qRT–PCR analysis. Midribs of the second to fifth leaves from the top were collected from 60-day-old old bm2-ref mutants and B73 wild-type plants and gene transcript abundance was determined. Values are means ±SE (n=3). (B) Schematic structure of MTHFR-1 cloned from the bm2-ref mutant. A 347 bp MITE insertion in the 5ʹUTR (–40 bp from the initial ATG start codon), a 414 bp deletion in the first intron (358–771 bp from the initial ATG start codon), and a single nucleotide transition (C→T) in the fourth exon (2308 bp from the initial ATG start codon) were identified in the MTHFR-1 of the bm2-ref mutant. (C) RT–PCR amplification of the ORF region of MTHFR-1 from the bm2-ref mutant and B73. (D) RT–PCR amplification of the 5ʹUTR and ORF regions of MTHFR-1 from the bm2-ref mutant and B73. (E) Folding structure of the MITE insertion predicted by the CentroidFold program. (F) Effects of the MITE insertion on transient transcriptional activity in maize leaf protoplasts. B73_5ʹNCR, 5ʹ-non-coding region (5ʹNCR) of MTHFR-1 from B73; bm2_5ʹNCR, 5ʹNCR of MTHFR-1 from the bm2-ref mutant; cB73_5ʹNCR, a chimeric 5ʹNCR including B73_5ʹNCR and the 347 bp MITE insertion recovered from the 5ʹUTR of the bm2-ref mutant. The ratio of firefly luciferase (LUC) to Renilla luciferase (REN) represents the activity of the 5ʹNCR of MTHFR-1. Values are means ±SE (n=3). Fig. 1. View largeDownload slide A MITE insertion in the 5ʹUTR reduced bm2 transcripts dramatically. (A) Relative transcript abundance of MTHFR-1 in the bm2-ref mutant and B73 (wild-type). Primers designed on the basis of the 5ʹUTR, ORF, and 3ʹUTR sequences were used in qRT–PCR analysis. Midribs of the second to fifth leaves from the top were collected from 60-day-old old bm2-ref mutants and B73 wild-type plants and gene transcript abundance was determined. Values are means ±SE (n=3). (B) Schematic structure of MTHFR-1 cloned from the bm2-ref mutant. A 347 bp MITE insertion in the 5ʹUTR (–40 bp from the initial ATG start codon), a 414 bp deletion in the first intron (358–771 bp from the initial ATG start codon), and a single nucleotide transition (C→T) in the fourth exon (2308 bp from the initial ATG start codon) were identified in the MTHFR-1 of the bm2-ref mutant. (C) RT–PCR amplification of the ORF region of MTHFR-1 from the bm2-ref mutant and B73. (D) RT–PCR amplification of the 5ʹUTR and ORF regions of MTHFR-1 from the bm2-ref mutant and B73. (E) Folding structure of the MITE insertion predicted by the CentroidFold program. (F) Effects of the MITE insertion on transient transcriptional activity in maize leaf protoplasts. B73_5ʹNCR, 5ʹ-non-coding region (5ʹNCR) of MTHFR-1 from B73; bm2_5ʹNCR, 5ʹNCR of MTHFR-1 from the bm2-ref mutant; cB73_5ʹNCR, a chimeric 5ʹNCR including B73_5ʹNCR and the 347 bp MITE insertion recovered from the 5ʹUTR of the bm2-ref mutant. The ratio of firefly luciferase (LUC) to Renilla luciferase (REN) represents the activity of the 5ʹNCR of MTHFR-1. Values are means ±SE (n=3). To find out whether the 347 bp MITE insertion in the 5ʹUTR can impair the expression of the gene, we cloned the 5ʹNCR of MTHFR-1, which included the 5ʹUTR and the 1.5 kb promoter region. Analysis of transient transcriptional activity in maize leaf protoplasts showed that the 5ʹNCR activity of MTHFR-1 from the bm2-ref mutant was 73% lower than that from B73 (Fig. 1F). To further confirm that the 347 bp MITE insertion was the cause of the low transcriptional activity of bm2_MTHFR-1, we inserted the 347 bp MITE into the 5ʹUTR of B73_MTHFR-1 at the same location as in the bm2-ref gene. As expected, the chimeric 5ʹNCR of cB73_MTHFR-1 showed a strong reduction in the signal intensity of luciferase, indicating severely impaired gene expression (Fig. 1F). Down-regulation of MTHFR-1 affected methyl metabolism and lignin biosynthesis Down-regulation of MTHFR-1 in the bm2-ref mutant caused an increase in the contents of 5,10-methylene-THF and THF, and a decrease in the 5-methyl-THF level, relative to their contents in the WT B73 (Fig. 2A, B). Since MTHFR is responsible for the conversion of 5,10-methylene-THF to 5-methyl-THF, it is likely that the increase in the 5,10-methylene-THF and THF content in the mutant is mostly due to an increase in 5,10-methylene-THF. In addition, the SAH and Hcy contents were significantly increased in the mutant, while the SAM and Met contents were unchanged (Fig. 2C, Supplementary Fig. S2). Thus, a significant increase in the ratio of SAH to SAM was observed in the mutant (Fig. 2D). Since 5-methyl-THF is required for the biosynthesis of Met from Hcy, one possible explanation for the observed effect on these metabolites is that the cells adjusted their biosynthetic machinery to maintain the levels of Met and SAM by increasing the de novo biosynthesis or reducing the turnover of Hcy, which is the direct precursor of Met. Fig. 2. View largeDownload slide Effect of MTHFR-1 down-regulation on methyl metabolites in maize. (A, B) Content of 5,10-methylene-THF and THF (A) and 5-methyl-THF (B) in bm2-ref mutants and B73 wild-type (WT) plants as measured by HPLC. (C) Content of SAM and SAH in bm2-ref and B73 as determined by ELISA. (D) Ratio of SAH to SAM in bm2-ref and B73. Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants. Values are means ±SE (n=6). Significant differences are indicated by asterisks: *P<0.05, **P<0.01 (Student’s t-test). FW, fresh weight. Fig. 2. View largeDownload slide Effect of MTHFR-1 down-regulation on methyl metabolites in maize. (A, B) Content of 5,10-methylene-THF and THF (A) and 5-methyl-THF (B) in bm2-ref mutants and B73 wild-type (WT) plants as measured by HPLC. (C) Content of SAM and SAH in bm2-ref and B73 as determined by ELISA. (D) Ratio of SAH to SAM in bm2-ref and B73. Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants. Values are means ±SE (n=6). Significant differences are indicated by asterisks: *P<0.05, **P<0.01 (Student’s t-test). FW, fresh weight. In addition, our results show that the bm2-ref mutant contains a similar amount of total lignin to B73 (Supplementary Table S3), as measured by the AcBr method. The lignin composition analysis using GC-MS revealed a dramatic decrease in G units in cell walls of the mutant. In contrast, no difference in S and 5-OH G units was determined between bm2-ref mutants and B73 WT plants (Supplementary Table S3). It is notable that we observed an apparently low S/G ratio (0.65) in B73 WT plants compared with the values (1.24–1.70) reported by Tang et al. (2014); this was because we used midribs rather than internodes for the lignin analysis. To assess the global effects of MTHFR-1 down-regulation on C1 and lignin metabolism, we examined gene expression in the bm2-ref mutant using microarray analysis. The most relevant genes among the 448 altered probe sets involved in C1 metabolism and monolignol biosynthesis are presented in Supplementary Table S4. Validation of the microarray results by qRT–PCR further revealed that the expression levels of METHIONINE SYNTHASE 1 and 2 (MS1 and MS2), HOMOCYSTEINE S-METHYLTRANSFERASE (HMT1), and ADENOSINE KINASE (ADK) were increased more than 3-fold in bm2-ref mutants compared with B73 WT plants, implying the presence of a coordinated network in C1 metabolism (Supplementary Table S5). The transcriptomic analysis thus showed that down-regulation of MTHFR-1 triggered up-regulation of the Met cycle genes. The 5-methyl-THF produced by MTHFR is a critical precursor for Met biosynthesis. The lower levels of 5-methyl-THF in the mutant, together with the finding that genes of the Met cycle are up-regulated, suggest that cells in the mutant attempt to compensate for the 5-methyl-THF deficiency by up-regulating the Met cycle. In contrast, the expression of genes related to monolignol biosynthesis was unchanged in the mutant (Supplementary Tables S4 and S5). Effects of SAM and SAH on enzymatic activities of O-methyltransferases in the lignin biosynthetic pathway To investigate the effects of SAM and SAH on the transmethylation of lignin intermediates, we first determined the activities of CCoAOMT and COMT in crude protein extracts from midribs of B73 WT plants against a range of SAM and SAH concentrations at a fixed concentration of caffeoyl CoA and 5-OH coniferyl alcohol, respectively. Our results showed that the activities of both CCoAOMT and COMT increased as the concentration of SAM was increased, until the enzymes were saturated (Supplementary Fig. S3). Neither CCoAOMT nor COMT exhibited substrate inhibition toward SAM (Supplementary Fig. S3). Furthermore, we studied the inhibition with SAH of the SAM-driven methylation reactions catalyzed by CCoAOMT and COMT in crude enzyme extracts prepared from B73 WT plants. Our results revealed a significant reduction in the catalytic activity of both CCoAOMT and COMT upon the addition of SAH (Fig. 3). Fig. 3. View largeDownload slide Activities of CCoAOMT and COMT at various SAM/SAH ratios. Crude enzyme extracts prepared from the midribs of B73 wild-type maize were assayed with 10 µM caffeoyl CoA and 10 µM 5-OH coniferyl alcohol, respectively, at different SAM and SAH concentrations. Fig. 3. View largeDownload slide Activities of CCoAOMT and COMT at various SAM/SAH ratios. Crude enzyme extracts prepared from the midribs of B73 wild-type maize were assayed with 10 µM caffeoyl CoA and 10 µM 5-OH coniferyl alcohol, respectively, at different SAM and SAH concentrations. To evaluate the affinities for SAM of CCoAOMT and COMT, we determined the Km values of the recombinant maize CCoAOMT2 (Zm00001d045206) and COMT (Zm00001d049541) proteins for SAM in vitro. Our results show that the recombinant CCoAOMT2 and COMT proteins have a comparable affinity for SAM (Table 1). Consistent with the findings in maize crude enzyme extracts, SAH inhibited catalytic activity of the recombinant maize CCoAOMT2 and COMT enzymes; however, CCoAOMT2 exhibited a lower Ki value for SAH than COMT (Table 1). Table 1. Kinetic analysis of recombinant OMT enzymes in the lignin biosynthetic pathway of maize OMT enzymes Km, SAM (μM) Vmax, SAM (μmol s–1 g protein–1) Kcat, SAM (min–1) Kcat/Km, SAM (μmol–1 min–1) Ki, SAH (µM) Mode of inhibition CCoAOMT 17.22 ± 0.82 2.95 ± 0.04 5.19 ± 0.07 0.30 1.89 ± 0.35 Competitive COMT 16.24 ± 1.68 1.47 ± 0.04 3.49 ± 0.10 0.21 5.26 ± 0.38 Competitive OMT enzymes Km, SAM (μM) Vmax, SAM (μmol s–1 g protein–1) Kcat, SAM (min–1) Kcat/Km, SAM (μmol–1 min–1) Ki, SAH (µM) Mode of inhibition CCoAOMT 17.22 ± 0.82 2.95 ± 0.04 5.19 ± 0.07 0.30 1.89 ± 0.35 Competitive COMT 16.24 ± 1.68 1.47 ± 0.04 3.49 ± 0.10 0.21 5.26 ± 0.38 Competitive Values are means ±SE (n=3). View Large Table 1. Kinetic analysis of recombinant OMT enzymes in the lignin biosynthetic pathway of maize OMT enzymes Km, SAM (μM) Vmax, SAM (μmol s–1 g protein–1) Kcat, SAM (min–1) Kcat/Km, SAM (μmol–1 min–1) Ki, SAH (µM) Mode of inhibition CCoAOMT 17.22 ± 0.82 2.95 ± 0.04 5.19 ± 0.07 0.30 1.89 ± 0.35 Competitive COMT 16.24 ± 1.68 1.47 ± 0.04 3.49 ± 0.10 0.21 5.26 ± 0.38 Competitive OMT enzymes Km, SAM (μM) Vmax, SAM (μmol s–1 g protein–1) Kcat, SAM (min–1) Kcat/Km, SAM (μmol–1 min–1) Ki, SAH (µM) Mode of inhibition CCoAOMT 17.22 ± 0.82 2.95 ± 0.04 5.19 ± 0.07 0.30 1.89 ± 0.35 Competitive COMT 16.24 ± 1.68 1.47 ± 0.04 3.49 ± 0.10 0.21 5.26 ± 0.38 Competitive Values are means ±SE (n=3). View Large Down-regulation of MTHFR-1 altered the phenylpropanoid profile of bm2 mutants Metabolite profiling performed by GC-MS revealed that 91 compounds accumulated differentially in the bm2-ref mutant compared with B73 (Fig. 4A, Supplementary Table S6). This method, however, was unsuitable for detecting C1 intermediates, owing to the low abundance and lability of these compounds in plant cells; thus we did not detect any C1 metabolites by GC-MS. In contrast, the bm2-ref mutants showed 5.5- and 3.4-fold increases in caffeic acid and p-coumaric acid contents, respectively, compared with B73 plants (Supplementary Table S6). Soluble phenylpropanoid profiling by reversed-phase liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry further revealed a similar increase in caffeoylquinic acid (peak 3) and p-coumaric acid (peak 4) in the mutant (Fig. 4B, C). Most importantly, we identified two novel metabolites (peaks 1 and 2) that were present in the mutant but absent in B73 (Fig. 4B, C). To determine whether these metabolites are directly derived from caffeyl alcohol, we first analyzed their UV-visible spectra. Our results were consistent with peaks 1 and 2 being the glucoside derivatives of hydroxycinnamyl alcohol (Supplementary Table S7). Fig. 4. View largeDownload slide Effects of MTHFR-1 down-regulation on phenylpropanoid accumulation in maize. (A) Volcano plot of 2-fold up- and down-regulated metabolites in the bm2-ref mutant. Means are shown (n=6). (B) Profile of soluble phenolics in methanolic extracts from midribs of bm2-ref mutants and B73 wild-type (WT) plants. The profile of soluble phenolics was performed by reversed-phase liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry. The greatly accumulated phenolics in the bm2 mutants were identified as caffeyl alcohol glucoside (peak 1), caffeyl alcohol acetyl glucoside (peak 2), caffeoylquinic acid (peak 3), and p-coumaric acid (peak 4) on the basis of their UV-visible spectra, mass spectra, and comparison with the authentic standard compounds. (C) Contents of the same four phenolics in bm2-ref mutants and B73 WT plants. Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref and B73 plants. DW, dry weight. Values are means ±SE (n=3). Fig. 4. View largeDownload slide Effects of MTHFR-1 down-regulation on phenylpropanoid accumulation in maize. (A) Volcano plot of 2-fold up- and down-regulated metabolites in the bm2-ref mutant. Means are shown (n=6). (B) Profile of soluble phenolics in methanolic extracts from midribs of bm2-ref mutants and B73 wild-type (WT) plants. The profile of soluble phenolics was performed by reversed-phase liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry. The greatly accumulated phenolics in the bm2 mutants were identified as caffeyl alcohol glucoside (peak 1), caffeyl alcohol acetyl glucoside (peak 2), caffeoylquinic acid (peak 3), and p-coumaric acid (peak 4) on the basis of their UV-visible spectra, mass spectra, and comparison with the authentic standard compounds. (C) Contents of the same four phenolics in bm2-ref mutants and B73 WT plants. Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref and B73 plants. DW, dry weight. Values are means ±SE (n=3). To elucidate the structure of the two novel compounds, the glucoside residues of phenolics were removed by enzymatic digestion. A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry analysis of β-glucosidase hydrolysis products and comparison with the authentic caffeyl alcohol standard confirmed that caffeyl alcohol is the aglycone of peaks 1 and 2 (Supplementary Table S7). Thus, peak 1 was identified as a glucoside derivative of caffeyl alcohol, and peak 2 as an acetyl glucoside derivative of caffeyl alcohol (Supplementary Table S7). Moreover, these glucoside derivatives of caffeyl alcohol were found to accumulate in the additional bm2-Mu mutants (Supplementary Fig. S4). Down-regulation of MTHFR-1 caused unusual C lignin accumulation in bm2 mutants To investigate whether the impaired 3-O-methylation pathway can lead to the integration of C lignin units into cell walls, we first conducted a detailed examination of the lignin composition. The characteristic ion peaks of the C, G, and S units were retrieved from the extracted ion chromatogram of thioacidolysis-derived lignin monomers of the bm2-ref mutant (Supplementary Fig. S5A). The extracted ion chromatogram of m/z 327 revealed a thioacidolysis-released lignin doublet present in the GC-MS profiles of midribs of bm2-ref mutants and B73 WT plants at the same retention time as the C lignins of Vanilla planifolia seed coats (Supplementary Fig. S5B). Moreover, lignin composition analysis showed that the bm2-ref mutants yielded 687.7% (8.35 ± 0.35 µmol g–1 CWR) more C units than the B73 WT plants (1.06 ± 0.18 µmol g–1 CWR) (Supplementary Fig. S5C). Next, 1H-13C 2D HSQC NMR was used to investigate the profiles of various lignin units in the cell walls of maize midribs (Fig. 5, Supplementary Table S8). The aliphatic regions of the 2D HSQC NMR spectrum revealed the typical β-O-4, β-β, β-5, and β-1 structures in lignin linkages of B73 and the bm2-ref mutant (Fig. 5A, B). Among these, the linear β-O-4 structure accounted for 48.8% of all lignin linkages in B73, whereas this type of linkage structure was substantially less common in bm2-ref. As a consequence, the proportion of condensed lignin linkages (β-β, β-5, and β-1) was significantly higher in the mutant (Fig. 5A, B). Furthermore, the aromatic subregions showed the signals for the conventional G/S/H lignin units in cell walls of B73 and bm2-ref (Fig. 5C, D). Most strikingly, the signals of unusual C lignin units were clearly observed in both B73 and bm2-ref. As anticipated, the fraction of C units in the bm2-ref mutant was notably higher than in B73 (Fig. 5C, D). In addition, the fraction of G units was significantly decreased in bm2-ref, whereas the fraction of S units was barely different between B73 and bm2-ref (Fig. 5C, D). These results are consistent with our thioacidolysis analysis and soluble phenolic profiling (Supplementary Tables S3 and S7; Fig. 4B, C). Fig. 5. View largeDownload slide 2D-NMR characterization of lignins extracted from midrib cell walls of bm2-ref mutants and B73 wild-type (WT) plants. The types of lignin linkages and proportions of lignin units were revealed in aliphatic and aromatic subregions of short-range 13C-1H correlation (HSQC) NMR spectra of B73 (A and C) and bm2 (B and D). Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants for analysis. H, p-hydroxyphenyl units; G, guaiacyl units; C, catechyl units; S, syringyl units. Fig. 5. View largeDownload slide 2D-NMR characterization of lignins extracted from midrib cell walls of bm2-ref mutants and B73 wild-type (WT) plants. The types of lignin linkages and proportions of lignin units were revealed in aliphatic and aromatic subregions of short-range 13C-1H correlation (HSQC) NMR spectra of B73 (A and C) and bm2 (B and D). Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants for analysis. H, p-hydroxyphenyl units; G, guaiacyl units; C, catechyl units; S, syringyl units. Saccharification efficiency of bm2 mutants The bm2-ref mutants maintained a dry matter biomass at the R1 stage (silk emergence) comparable to that of the B73 WT plants (Supplementary Fig. S6). Although no difference in AcBr lignin content was found between bm2-ref mutants and B73 WT plants, the dramatically altered lignin composition in the mutant (Supplementary Table S3) prompted us to investigate the saccharification efficiency of the biomass. The enzymatic hydrolysis efficiency of cell wall polysaccharides was 23.0% in B73 and 36.3% in bm2 mutant plants (a relative increase of 57.8%) (Supplementary Fig. S7). Discussion The maize bm2 mutant was first isolated in 1932 (Burnham and Brink 1932). Since then, it has been extensively investigated to characterize the effects of the mutant gene underlying the bm2 locus (Barrière et al., 2004; Shi et al., 2006). Recently, Tang et al. (2014) identified MTHFR-1 as the bm2 gene. In addition, the maize bm4 locus has been characterized and encodes a functional folylpolyglutamate synthase that locates directly upstream of MTHFR in the folate cycle (Li et al., 2015). These breakthrough studies suggested new targets that can be utilized for lignin bioengineering. However, the direct cause-and-effect relationship between methyl metabolism and lignin biosynthesis is not yet clearly elucidated from the context of bm2 mutants. The data we present here support a model in which reduced MTHFR expression leads to the accumulation of SAH without a change in the SAM content. Since SAH inhibits the COMT and CCoAOMT methyltransferases, this accumulation then slows down the methylation of lignin units and leads to unconventional C lignin accumulation in maize cell walls (Fig. 6). Moreover, our 2D HSQC NMR analysis clearly shows that the C units were deposited in cell walls of both B73 and bm2 plants. This is the first time that C lignins have been found in vegetative tissues other than seed coats (Chen et al., 2012a; Tobimatsu et al., 2013). Fig. 6. View largeDownload slide A model for the effects of down-regulation of MTHFR on methyl metabolism and lignin biosynthesis in plants. ADK, Adenosine kinase; AMP, adenosine monophosphate; CCoAOMT, caffeoyl CoA O-methyltransferase; COMT, caffeic acid O-methyltransferase; C lignin, catechyl lignin; FPGS, folylpolyglutamate synthetase; G lignin, guaiacyl lignin; 5-OH G lignin, 5-hydroxyl guaiacyl lignin; HMT, homocysteine S-methyltransferase; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine; SMM, S-methylmethionine; SAMS, S-adenosylmethionine synthase; THF, tetrahydrofolate; S lignin, syringyl lignin. Fig. 6. View largeDownload slide A model for the effects of down-regulation of MTHFR on methyl metabolism and lignin biosynthesis in plants. ADK, Adenosine kinase; AMP, adenosine monophosphate; CCoAOMT, caffeoyl CoA O-methyltransferase; COMT, caffeic acid O-methyltransferase; C lignin, catechyl lignin; FPGS, folylpolyglutamate synthetase; G lignin, guaiacyl lignin; 5-OH G lignin, 5-hydroxyl guaiacyl lignin; HMT, homocysteine S-methyltransferase; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine; SMM, S-methylmethionine; SAMS, S-adenosylmethionine synthase; THF, tetrahydrofolate; S lignin, syringyl lignin. MTHFRs are highly conserved from green algae to flowering plants. In this study, we cloned the full-length genomic sequence of MTHFR-1 from the bm2 mutant and recovered a MITE sequence from its 5ʹUTR. MITEs are small non-autonomous DNA transposons with short terminal inverted repeats and a high copy number in plant genomes, which participate in gene regulation through promoter enhancement/repression and exon/intron disruption besides the formation of genetic structure (Bureau and Wessler, 1994; Yang et al., 2005; Guillet-Claude et al., 2004). Our transient transcriptional activity assay further demonstrates that the MITE insertion in the 5ʹUTR of MTHFR-1 can substantially suppress gene transcription in maize. This result is in agreement with the observation that no green fluorescent protein signal is detected in Arabidopsis in the presence of an mPING MITE inserted between the 35S promoter and the translation start codon of green fluorescent protein (Yang et al., 2007). In addition, the potential disturbance of translation initiation caused by the MITE inversion might induce RNA decay in the bm2 mutant. The detailed mechanism of down-regulation of MTHFR-1 due to the MITE insertion remains unclear, but is worth investigating in the future. In addition, the 414 bp deletion in the intron of bm2-MTHFR did not result in any unusual RNA splicing. However, we could not rule out the possibility that this deletion might affect the expression levels of bm2-MTHFR. The plant MTHFR enzymes prefer NADH to NADPH as a reductant (Roje et al., 1999). Given the low ratio of NADH to NAD in plant cytosol, a reversible reaction catalyzed by NADH-dependent MTHFRs is expected to take place in plant cells (Roje et al., 1999). Our results indicate that down-regulation of MTHFR-1 in maize leads to a decrease in 5-methyl-THF and an increase in 5,10-methylene-THF and THF, suggesting that the forward MTHFR reaction is dominant in the plant cytosol of maize leaves under the tested growth conditions. Our results further suggest that the reduction in 5-methyl-THF impaired the remethylation of Hcy, causing an increase in the levels of both Hcy and SAH in the bm2 mutant. Recent studies have shown that mutations in this MTHFR and its upstream FPGS affect lignin biosynthesis in maize (Li et al., 2015; Tang et al., 2014). Thus, it was proposed that down-regulation of MTHFR in the bm2 mutant reduced lignin biosynthesis by causing a reduction in the level of SAM (Tang et al., 2014). Our results are not consistent with this proposed mechanism, as they predict that down-regulation of MTHFR in the bm2 mutant may reduce SAM accumulation. This finding is consistent, however, with observations in MTHFR-RNAi transgenic tobacco plants showing a similar lack of change in SAM levels (Hung et al., 2013). Our data suggest that down-regulation of MTHFR affects SAH rather than SAM levels in maize. Unlike SAM, SAH is fairly labile in tissue extracts, and therefore the levels of SAH are widely underestimated in plants (Hanson and Roje, 2001). Even given this poor detection situation, SAH was still up-regulated significantly in the bm2 mutant and exhibited dominant inhibition effects on the transmethylation reactions of lignin intermediates. The functions of lignin genes have yet to be widely characterized in monocot species as compared with dicot species. Examination of mutants deficient in lignin biosynthesis genes is needed to add the missing steps to the monocot lignin biosynthetic pathway. CCoAOMT is an important enzyme involved in the 3-O-methylation of both G and S lignins. Caffeoyl CoA has been designated as the substrate of CCoAOMT in vivo. Reduction of CCoAOMT activity in poplar and alfalfa reduces the rate of utilization of caffeoyl CoA, causing accumulation of the precursor in the form of caffeic acid glucoside through a feedback regulatory mechanism (Meyermans et al., 2000; Guo et al., 2001). Two CCoAOMTs with more than 90% amino acid identity are expressed at high levels in lignified tissues of maize (Vélez-Bermúdez et al., 2015) and therefore it is hard to obtain a CCoAOMT-deficient mutant with an obvious phenotype. Lignin composition analysis of the bm2 mutant indicates that down-regulation of MTHFR-1 affects the 3-O-methylation step in lignin biosynthesis, but not the 5-O-methylation step. This is consistent with our result showing that SAH inhibits CCoAOMT activity more strongly than COMT activity. The MTHFR-1 mutation, which dominantly affects the 3-O-methylation step mediated by CCoAOMT in lignin biosynthesis, also provides an ideal model to study the function of CCoAOMT in monocots. In contrast to what has been observed in poplar and alfalfa (Meyermans et al., 2000; Guo et al., 2001), disruption of the 3-O-methylation function in maize mainly shunted the redundant caffeoyl CoA toward caffeyl alcohol and led to a remarkable and unusual integration of C lignin into cell walls as a consequence of the reduction in G lignin. In addition, our finding is consistent with the observation that down-regulation of CCoAOMT led to the accumulation of C lignin units in Pinus radiata tracheary element cultures (Wagner et al., 2011). Thus, our results suggest that the biosynthesis of C and G lignins can be controlled by the fine-tuning of methyl metabolism in plants. The structure of the C lignins in the cell walls of bm2 mutants is currently unclear, so further investigation is needed to determine whether the C units are incorporated into the classical G-S lignins, or produce homopolymers as has been observed in the seed coats of V. planifolia (Chen et al., 2012a). Other metabolic fluxes induced in the bm2 mutant were the specific accumulation of caffeyl alcohol glucoside derivatives and a significant increase in caffeoylquinic acid and p-coumaric acid, which may reflect the impaired O-methylation reactions in lignin biosynthesis as well. Our data therefore suggest a complex coordinated regulatory network for the 3-O-methylation step in monocots. The approaches currently employed in lignin engineering are limited to the biosynthetic and regulatory genes of the lignin pathway. Crosstalk between the lignin and other biosynthetic pathways clearly exists, as evidenced by a finding that a loss-of-function mutation in the glucosamine-6-phosphate N-acetyltransferase gene leads to ectopic lignin accumulation (Nozaki et al., 2012). Our work shows that increasing the SAH content in maize leads to dramatically reduced biosynthesis of the G units, and incorporation of the C units into cell walls, but does not alter, or only slightly alters, the accumulation of S units and total lignin content. These changes in lignin biosynthesis can substantially improve the saccharification efficiency of cell walls. In addition, severe disturbance of lignin biosynthesis is usually associated with stunting of plant growth (Chapple et al., 1992). However, no growth deficiency was observed in the maize bm2-ref mutants. These results suggest that the moderate regulation of methyl metabolism genes, at least MTHFR, has no disadvantage for plant development and growth, which provides potential alternative strategies for cell wall engineering of biofuel and forage crops in the future. Supplementary data Supplementary data are available at JXB online. Fig. S1. MITE sequence inserted in the 5ʹ-non-coding region of the bm2 gene and its predicted folding structure. Fig. S2. Contents of Met (A) and Hcy (B) in bm2-ref mutants and B73 wild-type plants determined by ELISA. Fig. S3. Michaelis–Menten plots of the kinetic assays of maize CCoAOMT and COMT enzymes. Fig. S4. Contents of novel phenolics accumulated in bm2-Mu mutants. Fig. S5. Effects of MTHFR-1 down-regulation on lignin composition in maize. Fig. S6. Above-ground biomass of bm2-ref mutants and B73 wild-type plants. Fig. S7. Saccharification efficiency of stalk cell walls of bm2-ref mutants and B73 wild-type plants. Table S1. Primers used in this study. Table S2. Distribution of the MITE recovered from the 5ʹUTR of the bm2 gene in the maize genome. Table S3. Lignin content and composition of bm2-ref mutants and B73 wild-type plants. Table S4. Differentially expressed one-carbon metabolite and lignin biosynthesis genes found in the microarray analysis of bm2-ref mutants and B73 wild-type plants. Table S5. Verification of the altered expression level of C1 metabolism and lignin pathway genes in the bm2-ref mutant by qRT–PCR. Table S6. Differentially accumulated compounds in bm2-ref mutants and B73 wild-type plants. Table S7. Identification of soluble phenolics in methanolic extracts from the midribs of bm2 mutants. Table S8. Assignments of signals in 2D HSQC NMR spectra of lignin extracted from cell walls of bm2-ref mutants and B73 wild-type plants. Acknowledgements We thank Dr Richard A. Dixon for critical reading and discussion of the manuscript, and Cong Wang, Fali Bai, and Haiyan Yang in the public laboratory, QIBEBT, CAS, for assistance with LC-MS/MS and HPLC analysis. This research was supported by the National Key Technologies Research & Development Program—Seven Major Crops Breeding Project (grant no. 2016YFD0101803), the 100-Talent Program of the Chinese Academy of Sciences Foundation, and the Natural Science Foundation of China (grant no. 31470390). Abbreviations Abbreviations AcBr acetyl bromide bm2 brown midrib2 C catechyl C1 one-carbon CCoAOMT caffeoyl CoA O-methyltransferase COMT caffeic acid O-methyltransferase CWRs cell wall residues 2D HSQC NMR two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy G guaiacyl GC-MS gas chromatography-mass spectrometry H p-hydroxyphenyl Hcy homocysteine NCR non-coding region Met methionine MITE miniature inverted-repeat transposable element MTHFR methylenetetrahydrofolate reductase S syringyl SAH S-adenosyl homocysteine SAM S-adenosyl methionine THF tetrahydrofolate UTR untranslated region. References Ali F , Scott P , Bakht J , et al. 2010 . 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Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Methylenetetrahydrofolate reductase modulates methyl metabolism and lignin monomer methylation in maize

Journal of Experimental Botany , Volume Advance Article (16) – May 30, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com
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0022-0957
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1460-2431
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10.1093/jxb/ery208
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Abstract

Abstract The brown midrib2 (bm2) mutant of maize, which has a modified lignin composition, contains a mutation in the methylenetetrahydrofolate reductase (MTHFR) gene. Here, we show that a MITE transposon insertion caused down-regulation of MTHFR, with an accompanying decrease in 5-methyl-tetrahydrofolate and an increase in 5, 10-methylene-tetrahydrofolate and tetrahydrofolate in the bm2 mutant. Furthermore, MTHFR mutation did not change the content of S-adenosyl methionine (SAM), the methyl group donor involved in the biosynthesis of guaiacyl and syringyl lignins, but increased the level of S-adenosyl homocysteine (SAH), the demethylation product of SAM. Moreover, competitive inhibition of the maize caffeoyl CoA O-methyltransferase (CCoAOMT) and caffeic acid O-methyltransferase (COMT) enzyme activities by SAH was found, suggesting that the SAH/SAM ratio, rather than the concentration of SAM, regulates the transmethylation reactions of lignin intermediates. Phenolic profiling revealed that caffeoyl alcohol glucose derivatives accumulated in the bm2 mutant, indicating impaired 3-O-methylation of monolignols. A remarkable increase in the unusual catechyl lignin in the mutant demonstrates that MTHFR down-regulation mainly affects guaiacyl lignin biosynthesis, consistent with the observation that CCoAOMT is more sensitive to SAH inhibition than COMT. This study uncovered a novel regulatory mechanism in lignin biosynthesis, which may offer an effective approach to utilizing lignocellulosic feedstocks in the future. Lignin, maize, methylenetetrahydrofolate reductase, methyl metabolism, S-adenosyl homocysteine, S-adenosyl methionine Introduction Maize (Zea mays L.) originated in Central America and is one of the most widely grown crops in the world. Corn stover is a lignocellulosic feedstock that can be utilized to produce renewable energy. Brown midrib (bm) mutants of maize are spontaneous mutants that have reddish-brown vascular tissues in the leaf midrib and stalk. Among them, the bm3 mutant has been used in commercial hybrids because of its high cell wall digestibility (Sattler et al., 2010). Six bm mutants (bm1–6) have been identified in maize since the 1930s (Ali et al., 2010). To date, the bm1, 2, 3, and 4 loci have been identified to encode genes that can affect lignin biosynthesis (Vignols et al., 1995; Chen et al., 2012b; Tang et al., 2014; Li et al., 2015). Lignin is a complex phenolic polymer that contributes to structural support, water transport, and biotic and abiotic stress defense during plant growth and development (Boerjan et al., 2003). Examination of lignin-altered maize, switchgrass, Brachypodium, Arabidopsis, alfalfa, and poplar has shown that lignin modification can substantially improve forage digestibility, bioethanol production, and pulping efficiency (Pilate et al., 2002; Barrière et al., 2006; Chen and Dixon, 2007; Fu et al., 2011a, 2011b; Ho-Yue-Kuang et al., 2016; Smith et al., 2017). The monolignols, the lignin precursors, are synthesized through the hydroxylation and methoxylation of derivatives from the phenylpropanoid pathway. Three principal monolignols, named p-coumaryl, coniferyl, and sinapyl alcohols, are incorporated into lignin polymers to produce p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively (Boerjan et al., 2003). Their proportions vary among plant species and tissue types, but the abundance of G and S units is generally very high compared with the H unit (Chapple et al., 1992). Genetic manipulation of the ratio of G to S in the cell wall is complex and involves the regulation of both caffeoyl CoA O-methyltransferase (CCoAOMT) and caffeic acid O-methyltransferase (COMT) (Zhong et al., 1998; Li et al., 2000). The coordinated regulatory mechanism acting on the O-methyltransferases responsible for the biosynthesis of catechyl (C) and G lignins, however, still remains largely unknown. S-adenosyl methionine (SAM), as the primary methyl donor, is required for the biosynthesis of both G and S monolignols (Hanson et al., 2000), and therefore genetic regulation of SAM biosynthesis has the potential to reduce the extent of O-methylation of G and S monolignols (Shen et al., 2002). Previous studies in animals and microbes, however, have shown that S-adenosyl homocysteine (SAH), the demethylation product of SAM, can inhibit the enzymatic activities of methyltransferases by competing with SAM for the binding pocket (Keating et al., 1991; Nguyen et al., 2001). Thus, the ratio of SAH to SAM, rather than the level of SAM, is a crucial factor controlling the methylation status of these cells. However, the effects of SAM and SAH on methyltransferase activity have yet to be widely investigated in higher plants. The methyl moiety of SAM is derived from one-carbon (C1) metabolism and biosynthesized through folate-mediated reactions and the methionine (Met) cycle (Hanson et al., 2000; Hanson and Roje, 2001; Bradbury et al., 2014). C1 metabolism is an essential biological process in plants from green algae to angiosperms. The C1 units in different oxidation states (10-formyl-THF, 5-formyl-THF, 5,10-methenyl-THF, 5,10-methylene-THF, and 5-methyl-THF) are carried by tetrahydrofolate (THF). These C1 derivatives of THF are enzymatically interconverted between different oxidation states (Hanson and Roje, 2001). Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is required to convert 5,10-methylene-THF to 5-methyl-THF, which donates the methyl group to homocysteine (Hcy) for the synthesis of Met and then SAM (Roje et al., 1999). The vast majority of the flux of methyl groups through SAM in lignin-producing cells is used for the methylation of lignin monomers (Hanson and Roje, 2001). Although limited biochemical characterization of MTHFRs has previously been conducted with enzymes from Arabidopsis and maize, their detailed biological functions merit further investigation (Roje et al., 1999). Recently, an MTHFR-encoding gene (GRMZM2G347056, MTHFR-1) has been identified as the bm2 locus in maize (Tang et al., 2014). In the bm2 mutant, lignin accumulation is significantly reduced, and a brownish pigmentation first appears in the leaf midribs around the V4 stage (Tang et al., 2014). This mutation therefore points to the function of MTHFR in lignin biosynthesis. A predicted mechanism based on the possible effects of MTHFR on SAM biosynthesis is currently used to explain the impairment of lignin biosynthesis in the bm2 mutant (Tang et al., 2014), but in the absence of experimental evidence it remains largely unclear how MTHFR influences the flux of methyl groups towards the biosynthesis of monolignols and their derivatives. It has been shown, however, that regulation of MTHFR can affect the N-demethylation of nicotine markedly without significantly altering the contents of SAM and Met and without perturbing plant growth in transgenic tobacco plants in which MTHFR is down-regulated (Hung et al., 2013). To elucidate the function of MTHFR in methyl metabolism and lignin biosynthesis in maize, we cloned the full-length genomic sequence of MTHFR-1 from the bm2-ref mutant and recovered an insertion of a miniature inverted-repeat transposable element (MITE) from the 5ʹ untranslated region (5ʹUTR) that can cause a substantial decrease in MTHFR-1 transcript abundance. Our results showed that down-regulation of MTHFR-1 affected the accumulation of 5,10-methylene-THF and THF, 5-methyl-THF, SAH, and Hcy, but not Met and SAM, suggesting an important role of SAH in the regulation of transmethylation reactions of lignin intermediates. As a consequence, the O-methylation of lignin monomers was impaired, resulting in a significant reduction in G lignins and a remarkable accumulation of novel phenolics and unusual C lignins that derive from the polymerization of caffeyl alcohol in bm2 mutants; however, the content of S lignins was little changed. Furthermore, the altered lignin composition had no impact on total lignin content and plant growth, but led to a significant improvement in the efficiency of cell wall saccharification. Our findings suggest that MTHFR is a potential target for regulating the biosynthesis of G and C lignins, and may open up another avenue for lignin engineering of fuel and feed crops. Materials and methods Plant materials and growth conditions The maize stocks, 114A (bm2-PI586725), 134E (bm2-Mu-10-7061A), and 134K (bm2-Mu-10-7073G) containing the bm2 allele, were obtained from the Maize Genetics COOP Stock Center. The bm2-ref near-isogenic line, which was developed following six backcrosses of 114A with B73 as described by Vermerris et al. (2010), was used for functional characterization of MTHFR-1. The bm2-Mu mutant lines (134E and 134K) were used for soluble phenylpropanoid profiling analysis. Plant phenotype measurement was performed on maize plants grown in a greenhouse at 26 °C with 16 h light (390 µmol m–2 s–1)/8 h dark. Analysis of gene transcript abundance MTHFR-1 (GRMZM2G347056_V3, Zm00001d034602_V4) sequences were isolated from the bm2-ref mutant and the wild-type (WT) B73. Three primer pairs, designed in the 5ʹ untranslated region (UTR), open reading frame (ORF), and 3ʹUTR of MTHFR-1, were used to detect the expression level of MTHFR-1 in midribs by quantitative real-time reverse transcription–PCR (qRT–PCR) as described by Tang et al. (2014). The primers used for qRT–PCR are listed in Supplementary Table S1 at JXB online. Luciferase activity assay The 5ʹUTR and 1.5 kb promoter regions of MTHFR-1 were isolated from bm2-ref and B73. The 347 bp MITE insertion in the 5ʹUTR of MTHFR-1 was fused into the 5ʹUTR of MTHFR-1 from B73 at the same location as in the bm2-ref gene. The above three 5ʹ non-coding regions (NCRs) were cloned into the pGreenII 0800-LUC vector (Hellens et al., 2005), and the resulting constructs were transformed into maize leaf protoplasts as described by Sheen (1991). The relative ratio of firefly luciferase to Renilla luciferase was determined using a dual-luciferase reporter assay system (Promega). Determination of the intermediates in methyl metabolism Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants. The fresh samples were homogenized in liquid nitrogen and used for determination of the 5-methyl-THF and 5,10-methylene-THF contents by HPLC according to Hung et al. (2013). Immunoassay of Met, SAM, SAH, and Hcy was conducted as described by Hao et al. (2016). Microarray analysis and qRT–PCR validation The midribs were separated from the leaves of 60-day-old bm2-ref mutants and B73 WT plants. RNA extraction and purification, probe labeling, hybridization, and scanning for microarray analysis were conducted as previously described by Fu et al. (2012). The transcript abundance of differentially expressed probe sets related to C1 metabolism and the lignin pathway was validated by qRT–PCR. The primers used for qRT–PCR are listed in Supplementary Table S1. Assay of CCoAOMT and COMT activity Powdered fresh midrib tissues of B73 plants (~500 mg) were extracted for 3 h at 4 °C in extraction buffer (Liu et al., 2012). The samples were centrifuged at 17900 g for 20 min at 4 °C, and the extracts were desalted on PD-10 columns (Pharmacia). The ORF sequences of CCoAOMT2 (GRMZM2G099363_V3, Zm00001d045206_V4) and COMT (AC196475.3_FG004_V3, Zm00001d049541_V4) isolated from B73 were cloned into the pET28a vector to produce recombinant proteins in transformed Escherichia coli. Caffeoyl CoA and 5-OH coniferyl alcohol were used as substrates with crude enzyme extracts. The enzyme activity assay for CCoAOMT and COMT was performed as described by Liu et al. (2012). Biochemical characterization of the bm2 mutant Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants, homogenized in liquid nitrogen, and lyophilized. Polar and non-polar metabolite profiling of bm2-ref and B73 were performed by gas chromatography-mass spectrometry (GC-MS) as described by Broeckling et al. (2005). The methanolic extract from lyophilized materials including bm2-ref mutants, bm2-Mu mutants, and B73 WT plants was subjected to soluble phenolics profiling analysis by reversed-phase liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry (Fu et al., 2011b). The phenolic glucoside derivatives were degraded by β-glucosidase according to Tian and Dixon (2006) and its hydrolysis products were identified on the basis of their UV-visible spectra, mass spectra, and comparison with authentic standard compounds. Caffeyl alcohol and 5-OH coniferyl alcohol were synthesized by Chemistry Research Solution LLC (PA, USA), and p-coumaric acid was ordered from Sigma-Aldrich (St Louis, MO, USA). Lyophilized extractive free cell wall residues (CWRs) were used for lignin analysis. The acetyl bromide (AcBr) method (Hatfield et al., 1999) and the thioacidolysis method (Lapierre et al., 1995) were used to quantify the lignin content and composition of maize materials, respectively. Lignins were extracted from cell walls of bm2-ref and B73 plants as described by Funaoka and Fukatsu (1996), and were subjected to two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (2D HSQC NMR) analysis as described by Tobimatsu et al. (2013). Measurement of saccharification efficiency of cell walls Stalk samples were collected from bm2-ref mutants and B73 WT plants at the R1 stage (silk emergence) and dried in an oven at 40 °C for 1 week. Samples were ground through a Wiley mill with a 1 mm sieve, and the extractive free CWRs were used for saccharification efficiency analysis. Saccharification of the maize samples was performed following the analytical procedure described by Fu et al. (2011a). Briefly, solubilized sugars were yielded from CWRs digested by pretreatment with 1.5% H2SO4 at 121 °C for 40 min followed by washing with Milli-Q water and then exposure to a cellulase and cellobiase mixture for 72 h. The solubilized sugars were detected by the phenol–sulfuric acid assay method (Dubois et al., 1956). Statistical analysis Six biological replicates were used for analyzing the contents of Met, SAM, SAH, Hcy, and polar and non-polar metabolites in the bm2-ref mutant and B73 WT plants; for all other experiments, samples were collected from three biological replicates. The mean values were used for statistical analyses. Data from each trait were subjected to Student’s t-test. The significance of treatments was tested at the P=0.05 and 0.01 levels. Standard errors are provided in all tables and figures as appropriate. Results A MITE insertion reduced bm2 transcription efficiency Previous research has shown that the bm2 gene encodes a functional MTHFR (MTHFR-1) but its transcript abundance is substantially reduced in the mutant (Tang et al., 2014). To investigate the cause of the low MTHFR-1 transcript abundance, we generated the bm2-ref near-isogenic line in the B73 background after six backcrosses of 114A with B73. Our qRT–PCR analysis showed that the MTHFR-1 transcript abundance in the bm2-ref mutant was reduced to 1.4% of the control line value for the 5ʹ UTR and ~30% for the ORF and the 3ʹUTR (Fig. 1A). Furthermore, we isolated the MTHFR-1 genomic sequences from the bm2-ref mutant and WT (B73) maize. Two tandem duplications of the MITE insert were found in the first MTHFR-1 intron in the WT plants. A 414 bp deletion containing one of these duplications occurred in the same MTHFR-1 region in the bm2-ref mutant (Fig. 1B). Alignment between the full-length cDNA sequences of MTHFR-1 from the bm2-ref mutant and B73 revealed that the ORF sequence was the same except for a synonymous mutation (Fig. 1B). In addition to the deletion, a 347 bp insertion in the 5ʹUTR of MTHFR-1 was identified in the mutant (Fig. 1B–D, Supplementary Fig. S1). Bioinformatic analysis further revealed that the insertion sequence contains stem-loop structures and belongs to the Tourist-like MITE family, which may explain why 5ʹUTR transcripts of MTHFR-1 were barely detected in the bm2-ref mutant by qRT–PCR (Fig. 1E). Additionally, this type of MITE inserts only into introns and 3ʹUTRs, but not exons and 5ʹUTRs in the B73 maize genome (Supplementary Table S2). Fig. 1. View largeDownload slide A MITE insertion in the 5ʹUTR reduced bm2 transcripts dramatically. (A) Relative transcript abundance of MTHFR-1 in the bm2-ref mutant and B73 (wild-type). Primers designed on the basis of the 5ʹUTR, ORF, and 3ʹUTR sequences were used in qRT–PCR analysis. Midribs of the second to fifth leaves from the top were collected from 60-day-old old bm2-ref mutants and B73 wild-type plants and gene transcript abundance was determined. Values are means ±SE (n=3). (B) Schematic structure of MTHFR-1 cloned from the bm2-ref mutant. A 347 bp MITE insertion in the 5ʹUTR (–40 bp from the initial ATG start codon), a 414 bp deletion in the first intron (358–771 bp from the initial ATG start codon), and a single nucleotide transition (C→T) in the fourth exon (2308 bp from the initial ATG start codon) were identified in the MTHFR-1 of the bm2-ref mutant. (C) RT–PCR amplification of the ORF region of MTHFR-1 from the bm2-ref mutant and B73. (D) RT–PCR amplification of the 5ʹUTR and ORF regions of MTHFR-1 from the bm2-ref mutant and B73. (E) Folding structure of the MITE insertion predicted by the CentroidFold program. (F) Effects of the MITE insertion on transient transcriptional activity in maize leaf protoplasts. B73_5ʹNCR, 5ʹ-non-coding region (5ʹNCR) of MTHFR-1 from B73; bm2_5ʹNCR, 5ʹNCR of MTHFR-1 from the bm2-ref mutant; cB73_5ʹNCR, a chimeric 5ʹNCR including B73_5ʹNCR and the 347 bp MITE insertion recovered from the 5ʹUTR of the bm2-ref mutant. The ratio of firefly luciferase (LUC) to Renilla luciferase (REN) represents the activity of the 5ʹNCR of MTHFR-1. Values are means ±SE (n=3). Fig. 1. View largeDownload slide A MITE insertion in the 5ʹUTR reduced bm2 transcripts dramatically. (A) Relative transcript abundance of MTHFR-1 in the bm2-ref mutant and B73 (wild-type). Primers designed on the basis of the 5ʹUTR, ORF, and 3ʹUTR sequences were used in qRT–PCR analysis. Midribs of the second to fifth leaves from the top were collected from 60-day-old old bm2-ref mutants and B73 wild-type plants and gene transcript abundance was determined. Values are means ±SE (n=3). (B) Schematic structure of MTHFR-1 cloned from the bm2-ref mutant. A 347 bp MITE insertion in the 5ʹUTR (–40 bp from the initial ATG start codon), a 414 bp deletion in the first intron (358–771 bp from the initial ATG start codon), and a single nucleotide transition (C→T) in the fourth exon (2308 bp from the initial ATG start codon) were identified in the MTHFR-1 of the bm2-ref mutant. (C) RT–PCR amplification of the ORF region of MTHFR-1 from the bm2-ref mutant and B73. (D) RT–PCR amplification of the 5ʹUTR and ORF regions of MTHFR-1 from the bm2-ref mutant and B73. (E) Folding structure of the MITE insertion predicted by the CentroidFold program. (F) Effects of the MITE insertion on transient transcriptional activity in maize leaf protoplasts. B73_5ʹNCR, 5ʹ-non-coding region (5ʹNCR) of MTHFR-1 from B73; bm2_5ʹNCR, 5ʹNCR of MTHFR-1 from the bm2-ref mutant; cB73_5ʹNCR, a chimeric 5ʹNCR including B73_5ʹNCR and the 347 bp MITE insertion recovered from the 5ʹUTR of the bm2-ref mutant. The ratio of firefly luciferase (LUC) to Renilla luciferase (REN) represents the activity of the 5ʹNCR of MTHFR-1. Values are means ±SE (n=3). To find out whether the 347 bp MITE insertion in the 5ʹUTR can impair the expression of the gene, we cloned the 5ʹNCR of MTHFR-1, which included the 5ʹUTR and the 1.5 kb promoter region. Analysis of transient transcriptional activity in maize leaf protoplasts showed that the 5ʹNCR activity of MTHFR-1 from the bm2-ref mutant was 73% lower than that from B73 (Fig. 1F). To further confirm that the 347 bp MITE insertion was the cause of the low transcriptional activity of bm2_MTHFR-1, we inserted the 347 bp MITE into the 5ʹUTR of B73_MTHFR-1 at the same location as in the bm2-ref gene. As expected, the chimeric 5ʹNCR of cB73_MTHFR-1 showed a strong reduction in the signal intensity of luciferase, indicating severely impaired gene expression (Fig. 1F). Down-regulation of MTHFR-1 affected methyl metabolism and lignin biosynthesis Down-regulation of MTHFR-1 in the bm2-ref mutant caused an increase in the contents of 5,10-methylene-THF and THF, and a decrease in the 5-methyl-THF level, relative to their contents in the WT B73 (Fig. 2A, B). Since MTHFR is responsible for the conversion of 5,10-methylene-THF to 5-methyl-THF, it is likely that the increase in the 5,10-methylene-THF and THF content in the mutant is mostly due to an increase in 5,10-methylene-THF. In addition, the SAH and Hcy contents were significantly increased in the mutant, while the SAM and Met contents were unchanged (Fig. 2C, Supplementary Fig. S2). Thus, a significant increase in the ratio of SAH to SAM was observed in the mutant (Fig. 2D). Since 5-methyl-THF is required for the biosynthesis of Met from Hcy, one possible explanation for the observed effect on these metabolites is that the cells adjusted their biosynthetic machinery to maintain the levels of Met and SAM by increasing the de novo biosynthesis or reducing the turnover of Hcy, which is the direct precursor of Met. Fig. 2. View largeDownload slide Effect of MTHFR-1 down-regulation on methyl metabolites in maize. (A, B) Content of 5,10-methylene-THF and THF (A) and 5-methyl-THF (B) in bm2-ref mutants and B73 wild-type (WT) plants as measured by HPLC. (C) Content of SAM and SAH in bm2-ref and B73 as determined by ELISA. (D) Ratio of SAH to SAM in bm2-ref and B73. Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants. Values are means ±SE (n=6). Significant differences are indicated by asterisks: *P<0.05, **P<0.01 (Student’s t-test). FW, fresh weight. Fig. 2. View largeDownload slide Effect of MTHFR-1 down-regulation on methyl metabolites in maize. (A, B) Content of 5,10-methylene-THF and THF (A) and 5-methyl-THF (B) in bm2-ref mutants and B73 wild-type (WT) plants as measured by HPLC. (C) Content of SAM and SAH in bm2-ref and B73 as determined by ELISA. (D) Ratio of SAH to SAM in bm2-ref and B73. Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants. Values are means ±SE (n=6). Significant differences are indicated by asterisks: *P<0.05, **P<0.01 (Student’s t-test). FW, fresh weight. In addition, our results show that the bm2-ref mutant contains a similar amount of total lignin to B73 (Supplementary Table S3), as measured by the AcBr method. The lignin composition analysis using GC-MS revealed a dramatic decrease in G units in cell walls of the mutant. In contrast, no difference in S and 5-OH G units was determined between bm2-ref mutants and B73 WT plants (Supplementary Table S3). It is notable that we observed an apparently low S/G ratio (0.65) in B73 WT plants compared with the values (1.24–1.70) reported by Tang et al. (2014); this was because we used midribs rather than internodes for the lignin analysis. To assess the global effects of MTHFR-1 down-regulation on C1 and lignin metabolism, we examined gene expression in the bm2-ref mutant using microarray analysis. The most relevant genes among the 448 altered probe sets involved in C1 metabolism and monolignol biosynthesis are presented in Supplementary Table S4. Validation of the microarray results by qRT–PCR further revealed that the expression levels of METHIONINE SYNTHASE 1 and 2 (MS1 and MS2), HOMOCYSTEINE S-METHYLTRANSFERASE (HMT1), and ADENOSINE KINASE (ADK) were increased more than 3-fold in bm2-ref mutants compared with B73 WT plants, implying the presence of a coordinated network in C1 metabolism (Supplementary Table S5). The transcriptomic analysis thus showed that down-regulation of MTHFR-1 triggered up-regulation of the Met cycle genes. The 5-methyl-THF produced by MTHFR is a critical precursor for Met biosynthesis. The lower levels of 5-methyl-THF in the mutant, together with the finding that genes of the Met cycle are up-regulated, suggest that cells in the mutant attempt to compensate for the 5-methyl-THF deficiency by up-regulating the Met cycle. In contrast, the expression of genes related to monolignol biosynthesis was unchanged in the mutant (Supplementary Tables S4 and S5). Effects of SAM and SAH on enzymatic activities of O-methyltransferases in the lignin biosynthetic pathway To investigate the effects of SAM and SAH on the transmethylation of lignin intermediates, we first determined the activities of CCoAOMT and COMT in crude protein extracts from midribs of B73 WT plants against a range of SAM and SAH concentrations at a fixed concentration of caffeoyl CoA and 5-OH coniferyl alcohol, respectively. Our results showed that the activities of both CCoAOMT and COMT increased as the concentration of SAM was increased, until the enzymes were saturated (Supplementary Fig. S3). Neither CCoAOMT nor COMT exhibited substrate inhibition toward SAM (Supplementary Fig. S3). Furthermore, we studied the inhibition with SAH of the SAM-driven methylation reactions catalyzed by CCoAOMT and COMT in crude enzyme extracts prepared from B73 WT plants. Our results revealed a significant reduction in the catalytic activity of both CCoAOMT and COMT upon the addition of SAH (Fig. 3). Fig. 3. View largeDownload slide Activities of CCoAOMT and COMT at various SAM/SAH ratios. Crude enzyme extracts prepared from the midribs of B73 wild-type maize were assayed with 10 µM caffeoyl CoA and 10 µM 5-OH coniferyl alcohol, respectively, at different SAM and SAH concentrations. Fig. 3. View largeDownload slide Activities of CCoAOMT and COMT at various SAM/SAH ratios. Crude enzyme extracts prepared from the midribs of B73 wild-type maize were assayed with 10 µM caffeoyl CoA and 10 µM 5-OH coniferyl alcohol, respectively, at different SAM and SAH concentrations. To evaluate the affinities for SAM of CCoAOMT and COMT, we determined the Km values of the recombinant maize CCoAOMT2 (Zm00001d045206) and COMT (Zm00001d049541) proteins for SAM in vitro. Our results show that the recombinant CCoAOMT2 and COMT proteins have a comparable affinity for SAM (Table 1). Consistent with the findings in maize crude enzyme extracts, SAH inhibited catalytic activity of the recombinant maize CCoAOMT2 and COMT enzymes; however, CCoAOMT2 exhibited a lower Ki value for SAH than COMT (Table 1). Table 1. Kinetic analysis of recombinant OMT enzymes in the lignin biosynthetic pathway of maize OMT enzymes Km, SAM (μM) Vmax, SAM (μmol s–1 g protein–1) Kcat, SAM (min–1) Kcat/Km, SAM (μmol–1 min–1) Ki, SAH (µM) Mode of inhibition CCoAOMT 17.22 ± 0.82 2.95 ± 0.04 5.19 ± 0.07 0.30 1.89 ± 0.35 Competitive COMT 16.24 ± 1.68 1.47 ± 0.04 3.49 ± 0.10 0.21 5.26 ± 0.38 Competitive OMT enzymes Km, SAM (μM) Vmax, SAM (μmol s–1 g protein–1) Kcat, SAM (min–1) Kcat/Km, SAM (μmol–1 min–1) Ki, SAH (µM) Mode of inhibition CCoAOMT 17.22 ± 0.82 2.95 ± 0.04 5.19 ± 0.07 0.30 1.89 ± 0.35 Competitive COMT 16.24 ± 1.68 1.47 ± 0.04 3.49 ± 0.10 0.21 5.26 ± 0.38 Competitive Values are means ±SE (n=3). View Large Table 1. Kinetic analysis of recombinant OMT enzymes in the lignin biosynthetic pathway of maize OMT enzymes Km, SAM (μM) Vmax, SAM (μmol s–1 g protein–1) Kcat, SAM (min–1) Kcat/Km, SAM (μmol–1 min–1) Ki, SAH (µM) Mode of inhibition CCoAOMT 17.22 ± 0.82 2.95 ± 0.04 5.19 ± 0.07 0.30 1.89 ± 0.35 Competitive COMT 16.24 ± 1.68 1.47 ± 0.04 3.49 ± 0.10 0.21 5.26 ± 0.38 Competitive OMT enzymes Km, SAM (μM) Vmax, SAM (μmol s–1 g protein–1) Kcat, SAM (min–1) Kcat/Km, SAM (μmol–1 min–1) Ki, SAH (µM) Mode of inhibition CCoAOMT 17.22 ± 0.82 2.95 ± 0.04 5.19 ± 0.07 0.30 1.89 ± 0.35 Competitive COMT 16.24 ± 1.68 1.47 ± 0.04 3.49 ± 0.10 0.21 5.26 ± 0.38 Competitive Values are means ±SE (n=3). View Large Down-regulation of MTHFR-1 altered the phenylpropanoid profile of bm2 mutants Metabolite profiling performed by GC-MS revealed that 91 compounds accumulated differentially in the bm2-ref mutant compared with B73 (Fig. 4A, Supplementary Table S6). This method, however, was unsuitable for detecting C1 intermediates, owing to the low abundance and lability of these compounds in plant cells; thus we did not detect any C1 metabolites by GC-MS. In contrast, the bm2-ref mutants showed 5.5- and 3.4-fold increases in caffeic acid and p-coumaric acid contents, respectively, compared with B73 plants (Supplementary Table S6). Soluble phenylpropanoid profiling by reversed-phase liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry further revealed a similar increase in caffeoylquinic acid (peak 3) and p-coumaric acid (peak 4) in the mutant (Fig. 4B, C). Most importantly, we identified two novel metabolites (peaks 1 and 2) that were present in the mutant but absent in B73 (Fig. 4B, C). To determine whether these metabolites are directly derived from caffeyl alcohol, we first analyzed their UV-visible spectra. Our results were consistent with peaks 1 and 2 being the glucoside derivatives of hydroxycinnamyl alcohol (Supplementary Table S7). Fig. 4. View largeDownload slide Effects of MTHFR-1 down-regulation on phenylpropanoid accumulation in maize. (A) Volcano plot of 2-fold up- and down-regulated metabolites in the bm2-ref mutant. Means are shown (n=6). (B) Profile of soluble phenolics in methanolic extracts from midribs of bm2-ref mutants and B73 wild-type (WT) plants. The profile of soluble phenolics was performed by reversed-phase liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry. The greatly accumulated phenolics in the bm2 mutants were identified as caffeyl alcohol glucoside (peak 1), caffeyl alcohol acetyl glucoside (peak 2), caffeoylquinic acid (peak 3), and p-coumaric acid (peak 4) on the basis of their UV-visible spectra, mass spectra, and comparison with the authentic standard compounds. (C) Contents of the same four phenolics in bm2-ref mutants and B73 WT plants. Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref and B73 plants. DW, dry weight. Values are means ±SE (n=3). Fig. 4. View largeDownload slide Effects of MTHFR-1 down-regulation on phenylpropanoid accumulation in maize. (A) Volcano plot of 2-fold up- and down-regulated metabolites in the bm2-ref mutant. Means are shown (n=6). (B) Profile of soluble phenolics in methanolic extracts from midribs of bm2-ref mutants and B73 wild-type (WT) plants. The profile of soluble phenolics was performed by reversed-phase liquid chromatography coupled with photodiode array detection and electrospray ionization tandem mass spectrometry. The greatly accumulated phenolics in the bm2 mutants were identified as caffeyl alcohol glucoside (peak 1), caffeyl alcohol acetyl glucoside (peak 2), caffeoylquinic acid (peak 3), and p-coumaric acid (peak 4) on the basis of their UV-visible spectra, mass spectra, and comparison with the authentic standard compounds. (C) Contents of the same four phenolics in bm2-ref mutants and B73 WT plants. Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref and B73 plants. DW, dry weight. Values are means ±SE (n=3). To elucidate the structure of the two novel compounds, the glucoside residues of phenolics were removed by enzymatic digestion. A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry analysis of β-glucosidase hydrolysis products and comparison with the authentic caffeyl alcohol standard confirmed that caffeyl alcohol is the aglycone of peaks 1 and 2 (Supplementary Table S7). Thus, peak 1 was identified as a glucoside derivative of caffeyl alcohol, and peak 2 as an acetyl glucoside derivative of caffeyl alcohol (Supplementary Table S7). Moreover, these glucoside derivatives of caffeyl alcohol were found to accumulate in the additional bm2-Mu mutants (Supplementary Fig. S4). Down-regulation of MTHFR-1 caused unusual C lignin accumulation in bm2 mutants To investigate whether the impaired 3-O-methylation pathway can lead to the integration of C lignin units into cell walls, we first conducted a detailed examination of the lignin composition. The characteristic ion peaks of the C, G, and S units were retrieved from the extracted ion chromatogram of thioacidolysis-derived lignin monomers of the bm2-ref mutant (Supplementary Fig. S5A). The extracted ion chromatogram of m/z 327 revealed a thioacidolysis-released lignin doublet present in the GC-MS profiles of midribs of bm2-ref mutants and B73 WT plants at the same retention time as the C lignins of Vanilla planifolia seed coats (Supplementary Fig. S5B). Moreover, lignin composition analysis showed that the bm2-ref mutants yielded 687.7% (8.35 ± 0.35 µmol g–1 CWR) more C units than the B73 WT plants (1.06 ± 0.18 µmol g–1 CWR) (Supplementary Fig. S5C). Next, 1H-13C 2D HSQC NMR was used to investigate the profiles of various lignin units in the cell walls of maize midribs (Fig. 5, Supplementary Table S8). The aliphatic regions of the 2D HSQC NMR spectrum revealed the typical β-O-4, β-β, β-5, and β-1 structures in lignin linkages of B73 and the bm2-ref mutant (Fig. 5A, B). Among these, the linear β-O-4 structure accounted for 48.8% of all lignin linkages in B73, whereas this type of linkage structure was substantially less common in bm2-ref. As a consequence, the proportion of condensed lignin linkages (β-β, β-5, and β-1) was significantly higher in the mutant (Fig. 5A, B). Furthermore, the aromatic subregions showed the signals for the conventional G/S/H lignin units in cell walls of B73 and bm2-ref (Fig. 5C, D). Most strikingly, the signals of unusual C lignin units were clearly observed in both B73 and bm2-ref. As anticipated, the fraction of C units in the bm2-ref mutant was notably higher than in B73 (Fig. 5C, D). In addition, the fraction of G units was significantly decreased in bm2-ref, whereas the fraction of S units was barely different between B73 and bm2-ref (Fig. 5C, D). These results are consistent with our thioacidolysis analysis and soluble phenolic profiling (Supplementary Tables S3 and S7; Fig. 4B, C). Fig. 5. View largeDownload slide 2D-NMR characterization of lignins extracted from midrib cell walls of bm2-ref mutants and B73 wild-type (WT) plants. The types of lignin linkages and proportions of lignin units were revealed in aliphatic and aromatic subregions of short-range 13C-1H correlation (HSQC) NMR spectra of B73 (A and C) and bm2 (B and D). Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants for analysis. H, p-hydroxyphenyl units; G, guaiacyl units; C, catechyl units; S, syringyl units. Fig. 5. View largeDownload slide 2D-NMR characterization of lignins extracted from midrib cell walls of bm2-ref mutants and B73 wild-type (WT) plants. The types of lignin linkages and proportions of lignin units were revealed in aliphatic and aromatic subregions of short-range 13C-1H correlation (HSQC) NMR spectra of B73 (A and C) and bm2 (B and D). Midribs of the second to fifth leaves from the top were collected from 60-day-old bm2-ref mutants and B73 WT plants for analysis. H, p-hydroxyphenyl units; G, guaiacyl units; C, catechyl units; S, syringyl units. Saccharification efficiency of bm2 mutants The bm2-ref mutants maintained a dry matter biomass at the R1 stage (silk emergence) comparable to that of the B73 WT plants (Supplementary Fig. S6). Although no difference in AcBr lignin content was found between bm2-ref mutants and B73 WT plants, the dramatically altered lignin composition in the mutant (Supplementary Table S3) prompted us to investigate the saccharification efficiency of the biomass. The enzymatic hydrolysis efficiency of cell wall polysaccharides was 23.0% in B73 and 36.3% in bm2 mutant plants (a relative increase of 57.8%) (Supplementary Fig. S7). Discussion The maize bm2 mutant was first isolated in 1932 (Burnham and Brink 1932). Since then, it has been extensively investigated to characterize the effects of the mutant gene underlying the bm2 locus (Barrière et al., 2004; Shi et al., 2006). Recently, Tang et al. (2014) identified MTHFR-1 as the bm2 gene. In addition, the maize bm4 locus has been characterized and encodes a functional folylpolyglutamate synthase that locates directly upstream of MTHFR in the folate cycle (Li et al., 2015). These breakthrough studies suggested new targets that can be utilized for lignin bioengineering. However, the direct cause-and-effect relationship between methyl metabolism and lignin biosynthesis is not yet clearly elucidated from the context of bm2 mutants. The data we present here support a model in which reduced MTHFR expression leads to the accumulation of SAH without a change in the SAM content. Since SAH inhibits the COMT and CCoAOMT methyltransferases, this accumulation then slows down the methylation of lignin units and leads to unconventional C lignin accumulation in maize cell walls (Fig. 6). Moreover, our 2D HSQC NMR analysis clearly shows that the C units were deposited in cell walls of both B73 and bm2 plants. This is the first time that C lignins have been found in vegetative tissues other than seed coats (Chen et al., 2012a; Tobimatsu et al., 2013). Fig. 6. View largeDownload slide A model for the effects of down-regulation of MTHFR on methyl metabolism and lignin biosynthesis in plants. ADK, Adenosine kinase; AMP, adenosine monophosphate; CCoAOMT, caffeoyl CoA O-methyltransferase; COMT, caffeic acid O-methyltransferase; C lignin, catechyl lignin; FPGS, folylpolyglutamate synthetase; G lignin, guaiacyl lignin; 5-OH G lignin, 5-hydroxyl guaiacyl lignin; HMT, homocysteine S-methyltransferase; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine; SMM, S-methylmethionine; SAMS, S-adenosylmethionine synthase; THF, tetrahydrofolate; S lignin, syringyl lignin. Fig. 6. View largeDownload slide A model for the effects of down-regulation of MTHFR on methyl metabolism and lignin biosynthesis in plants. ADK, Adenosine kinase; AMP, adenosine monophosphate; CCoAOMT, caffeoyl CoA O-methyltransferase; COMT, caffeic acid O-methyltransferase; C lignin, catechyl lignin; FPGS, folylpolyglutamate synthetase; G lignin, guaiacyl lignin; 5-OH G lignin, 5-hydroxyl guaiacyl lignin; HMT, homocysteine S-methyltransferase; MS, methionine synthase; MTHFR, methylenetetrahydrofolate reductase; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine; SMM, S-methylmethionine; SAMS, S-adenosylmethionine synthase; THF, tetrahydrofolate; S lignin, syringyl lignin. MTHFRs are highly conserved from green algae to flowering plants. In this study, we cloned the full-length genomic sequence of MTHFR-1 from the bm2 mutant and recovered a MITE sequence from its 5ʹUTR. MITEs are small non-autonomous DNA transposons with short terminal inverted repeats and a high copy number in plant genomes, which participate in gene regulation through promoter enhancement/repression and exon/intron disruption besides the formation of genetic structure (Bureau and Wessler, 1994; Yang et al., 2005; Guillet-Claude et al., 2004). Our transient transcriptional activity assay further demonstrates that the MITE insertion in the 5ʹUTR of MTHFR-1 can substantially suppress gene transcription in maize. This result is in agreement with the observation that no green fluorescent protein signal is detected in Arabidopsis in the presence of an mPING MITE inserted between the 35S promoter and the translation start codon of green fluorescent protein (Yang et al., 2007). In addition, the potential disturbance of translation initiation caused by the MITE inversion might induce RNA decay in the bm2 mutant. The detailed mechanism of down-regulation of MTHFR-1 due to the MITE insertion remains unclear, but is worth investigating in the future. In addition, the 414 bp deletion in the intron of bm2-MTHFR did not result in any unusual RNA splicing. However, we could not rule out the possibility that this deletion might affect the expression levels of bm2-MTHFR. The plant MTHFR enzymes prefer NADH to NADPH as a reductant (Roje et al., 1999). Given the low ratio of NADH to NAD in plant cytosol, a reversible reaction catalyzed by NADH-dependent MTHFRs is expected to take place in plant cells (Roje et al., 1999). Our results indicate that down-regulation of MTHFR-1 in maize leads to a decrease in 5-methyl-THF and an increase in 5,10-methylene-THF and THF, suggesting that the forward MTHFR reaction is dominant in the plant cytosol of maize leaves under the tested growth conditions. Our results further suggest that the reduction in 5-methyl-THF impaired the remethylation of Hcy, causing an increase in the levels of both Hcy and SAH in the bm2 mutant. Recent studies have shown that mutations in this MTHFR and its upstream FPGS affect lignin biosynthesis in maize (Li et al., 2015; Tang et al., 2014). Thus, it was proposed that down-regulation of MTHFR in the bm2 mutant reduced lignin biosynthesis by causing a reduction in the level of SAM (Tang et al., 2014). Our results are not consistent with this proposed mechanism, as they predict that down-regulation of MTHFR in the bm2 mutant may reduce SAM accumulation. This finding is consistent, however, with observations in MTHFR-RNAi transgenic tobacco plants showing a similar lack of change in SAM levels (Hung et al., 2013). Our data suggest that down-regulation of MTHFR affects SAH rather than SAM levels in maize. Unlike SAM, SAH is fairly labile in tissue extracts, and therefore the levels of SAH are widely underestimated in plants (Hanson and Roje, 2001). Even given this poor detection situation, SAH was still up-regulated significantly in the bm2 mutant and exhibited dominant inhibition effects on the transmethylation reactions of lignin intermediates. The functions of lignin genes have yet to be widely characterized in monocot species as compared with dicot species. Examination of mutants deficient in lignin biosynthesis genes is needed to add the missing steps to the monocot lignin biosynthetic pathway. CCoAOMT is an important enzyme involved in the 3-O-methylation of both G and S lignins. Caffeoyl CoA has been designated as the substrate of CCoAOMT in vivo. Reduction of CCoAOMT activity in poplar and alfalfa reduces the rate of utilization of caffeoyl CoA, causing accumulation of the precursor in the form of caffeic acid glucoside through a feedback regulatory mechanism (Meyermans et al., 2000; Guo et al., 2001). Two CCoAOMTs with more than 90% amino acid identity are expressed at high levels in lignified tissues of maize (Vélez-Bermúdez et al., 2015) and therefore it is hard to obtain a CCoAOMT-deficient mutant with an obvious phenotype. Lignin composition analysis of the bm2 mutant indicates that down-regulation of MTHFR-1 affects the 3-O-methylation step in lignin biosynthesis, but not the 5-O-methylation step. This is consistent with our result showing that SAH inhibits CCoAOMT activity more strongly than COMT activity. The MTHFR-1 mutation, which dominantly affects the 3-O-methylation step mediated by CCoAOMT in lignin biosynthesis, also provides an ideal model to study the function of CCoAOMT in monocots. In contrast to what has been observed in poplar and alfalfa (Meyermans et al., 2000; Guo et al., 2001), disruption of the 3-O-methylation function in maize mainly shunted the redundant caffeoyl CoA toward caffeyl alcohol and led to a remarkable and unusual integration of C lignin into cell walls as a consequence of the reduction in G lignin. In addition, our finding is consistent with the observation that down-regulation of CCoAOMT led to the accumulation of C lignin units in Pinus radiata tracheary element cultures (Wagner et al., 2011). Thus, our results suggest that the biosynthesis of C and G lignins can be controlled by the fine-tuning of methyl metabolism in plants. The structure of the C lignins in the cell walls of bm2 mutants is currently unclear, so further investigation is needed to determine whether the C units are incorporated into the classical G-S lignins, or produce homopolymers as has been observed in the seed coats of V. planifolia (Chen et al., 2012a). Other metabolic fluxes induced in the bm2 mutant were the specific accumulation of caffeyl alcohol glucoside derivatives and a significant increase in caffeoylquinic acid and p-coumaric acid, which may reflect the impaired O-methylation reactions in lignin biosynthesis as well. Our data therefore suggest a complex coordinated regulatory network for the 3-O-methylation step in monocots. The approaches currently employed in lignin engineering are limited to the biosynthetic and regulatory genes of the lignin pathway. Crosstalk between the lignin and other biosynthetic pathways clearly exists, as evidenced by a finding that a loss-of-function mutation in the glucosamine-6-phosphate N-acetyltransferase gene leads to ectopic lignin accumulation (Nozaki et al., 2012). Our work shows that increasing the SAH content in maize leads to dramatically reduced biosynthesis of the G units, and incorporation of the C units into cell walls, but does not alter, or only slightly alters, the accumulation of S units and total lignin content. These changes in lignin biosynthesis can substantially improve the saccharification efficiency of cell walls. In addition, severe disturbance of lignin biosynthesis is usually associated with stunting of plant growth (Chapple et al., 1992). However, no growth deficiency was observed in the maize bm2-ref mutants. These results suggest that the moderate regulation of methyl metabolism genes, at least MTHFR, has no disadvantage for plant development and growth, which provides potential alternative strategies for cell wall engineering of biofuel and forage crops in the future. Supplementary data Supplementary data are available at JXB online. Fig. S1. MITE sequence inserted in the 5ʹ-non-coding region of the bm2 gene and its predicted folding structure. Fig. S2. Contents of Met (A) and Hcy (B) in bm2-ref mutants and B73 wild-type plants determined by ELISA. Fig. S3. Michaelis–Menten plots of the kinetic assays of maize CCoAOMT and COMT enzymes. Fig. S4. Contents of novel phenolics accumulated in bm2-Mu mutants. Fig. S5. Effects of MTHFR-1 down-regulation on lignin composition in maize. Fig. S6. Above-ground biomass of bm2-ref mutants and B73 wild-type plants. Fig. S7. Saccharification efficiency of stalk cell walls of bm2-ref mutants and B73 wild-type plants. Table S1. Primers used in this study. Table S2. Distribution of the MITE recovered from the 5ʹUTR of the bm2 gene in the maize genome. Table S3. Lignin content and composition of bm2-ref mutants and B73 wild-type plants. Table S4. Differentially expressed one-carbon metabolite and lignin biosynthesis genes found in the microarray analysis of bm2-ref mutants and B73 wild-type plants. Table S5. Verification of the altered expression level of C1 metabolism and lignin pathway genes in the bm2-ref mutant by qRT–PCR. Table S6. Differentially accumulated compounds in bm2-ref mutants and B73 wild-type plants. Table S7. Identification of soluble phenolics in methanolic extracts from the midribs of bm2 mutants. Table S8. Assignments of signals in 2D HSQC NMR spectra of lignin extracted from cell walls of bm2-ref mutants and B73 wild-type plants. Acknowledgements We thank Dr Richard A. Dixon for critical reading and discussion of the manuscript, and Cong Wang, Fali Bai, and Haiyan Yang in the public laboratory, QIBEBT, CAS, for assistance with LC-MS/MS and HPLC analysis. This research was supported by the National Key Technologies Research & Development Program—Seven Major Crops Breeding Project (grant no. 2016YFD0101803), the 100-Talent Program of the Chinese Academy of Sciences Foundation, and the Natural Science Foundation of China (grant no. 31470390). Abbreviations Abbreviations AcBr acetyl bromide bm2 brown midrib2 C catechyl C1 one-carbon CCoAOMT caffeoyl CoA O-methyltransferase COMT caffeic acid O-methyltransferase CWRs cell wall residues 2D HSQC NMR two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy G guaiacyl GC-MS gas chromatography-mass spectrometry H p-hydroxyphenyl Hcy homocysteine NCR non-coding region Met methionine MITE miniature inverted-repeat transposable element MTHFR methylenetetrahydrofolate reductase S syringyl SAH S-adenosyl homocysteine SAM S-adenosyl methionine THF tetrahydrofolate UTR untranslated region. References Ali F , Scott P , Bakht J , et al. 2010 . 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Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

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Journal of Experimental BotanyOxford University Press

Published: May 30, 2018

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