Please wait a minute...
Shopping Cart Email List Chinese
Advanced Search Fig/Tab
Frontiers of Agricultural Science and Engineering

ISSN 2095-7505

ISSN 2095-977X(Online)

CN 10-1204/S

Postal Subscription Code 80-906

Featured Articles  |  Most Downloaded  |  Most Reading
Online Submission Subscribe to Our RSS Content Feed
Front. Agr. Sci. Eng.    2018, Vol. 5 Issue (1) : 98-107    https://doi.org/10.15302/J-FASE-2017198
RESEARCH ARTICLE
Functional characterization of caffeic acid O-methyltransferase in internode lignification of switchgrass (Panicum virgatum)
Fengyan WU1,2, Zhenying WU1, Aiguo YANG2, Shanshan JIANG1, Zeng-Yu WANG3, Chunxiang FU1,3()
1. Key Laboratory of Energy Genetics of Shandong Province/Qingdao Engineering Research Center of Biomass Resources and Environment/Key Laboratory of Biofuels/Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, China
2. Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, China
3. Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore, OK73401, USA
 Download: PDF(1713 KB)   HTML
 Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
Abstract

Caffeic acid O-methyltransferase (COMT) is a crucial enzyme that mainly methylates phenylpropanoid meta-hydroxyl of C5 in the biosynthesis of syringyl lignin in angiosperms. A putative COMT, named as PvCOMT1, was isolated from switchgrass (Panicum virgatum), a C4 warm-season dual-purpose forage and bioenergy crop. Our results showed that recombinant PvCOMT1 enzyme protein catalyzed the methylation of 5-OH coniferyl alcohol, 5-OH coniferaldehyde (CAld5H) and 5-OH ferulic acid. Further in vitro studies indicate that CAld5H can dominate COMT-mediated reactions by inhibiting the methylation of the other substrates. Transgenic switchgrass plants generated by an RNAi approach were further employed to study the function of COMT in internode lignification. A dramatic decrease in syringyl lignin units coupled with an obvious incorporation in 5-OH guaiacyl lignin units were observed in the COMT-RNAi transgenic plants. However, the constitutive suppression of COMT in switchgrass plants altered neither the pattern of lignin deposition along the stem nor the anatomical structure of internodes. Consistent with the biochemical characterization of PvCOMT1, a significant decrease in sinapaldehyde was found in the COMT-RNAi transgenic switchgrass plants, suggesting that CAld5H could be the optimal intermediate in the biosynthesis syringyl lignin.
Keywords biofuel crop      caffeic acid O-methyltransferase      forage      lignin      Panicum virgatum      switchgrass      transgenic plant     
Corresponding Author(s): Chunxiang FU   
Just Accepted Date: 27 December 2017   Online First Date: 08 February 2018    Issue Date: 21 March 2018
 Cite this article:   
Fengyan WU,Zhenying WU,Aiguo YANG, et al. Functional characterization of caffeic acid O-methyltransferase in internode lignification of switchgrass (Panicum virgatum)[J]. Front. Agr. Sci. Eng. , 2018, 5(1): 98-107.
 URL:  
https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2017198
https://academic.hep.com.cn/fase/EN/Y2018/V5/I1/98
Fig.1  Identification of caffeic acid O-methyltransferases from the switchgrass genome. (a) Phylogenetic tree analysis of plant COMT (caffeic acid O-methyltransferase) and CCoAOMT (caffeoyl CoA O-methyltransferase) protein sequences. Switchgrass PvCOMTs and other members of the O-methyltransferases (OMT) family in Arabidopsis thaliana, Medicago truncatula, Nicotiana tabacum, Populus trichocarpa, Brachypodium distachyon, Festuca arundinacea, Festuca tabacum, Lolium perenne, Miscanthus sinensis, Oryza sativa, Saccharum officinarum and Zea mays. GenBank accession numbers are shown after species names. Phylogenetic tree of deduced OMT amino acid sequences constructed by using the neighbor-joining method. Bootstrap values (%) based on 1000 replications are indicated at nodes; (b) gene expression analysis of PvCOMT1 and PvCOMT2. The data was downloaded from Switchgrass Functional Genomics Server.
Fig.2  Effects of 5-OH coniferaldehyde on the O-methyltransferase activity of recombinant PvCOMT (a) and extractable protein from switchgrass internodes (b). CAld5H, 5-OH coniferaldehyde; CAlc5H, 5-OH coniferyl alcohol; FAc5H, 5-OH ferulic acid. Values are means±SE (n = 3).
Fig.3  Mäule staining of internodes cross sections (basal to distal) of the stems of control (a–d) and transgenic (e–h) switchgrass plants. Bar= 0.1 mm.
Fig.4  Lignification pattern of internodes in control and transgenic switchgrass plants. Yields of syringyl (S) lignin (a) and 5-OH guaiacyl (G) lignin (b) units determined by gas chromatography-mass spectrometry. Values are means±SE (n = 3).
Fig.5  Microarray analysis of caffeic acid O-methyltransferase (COMT)-RNAi transgenic switchgrass plants. (a) Transcript abundance of COMT gene in different internodes of transgenic plants revealed by qRT-PCR. Switchgrass Ubq1 was used as the reference for normalization. Values are means±SE (n = 3); (b) the number of altered probe sets in microarray chips; (c) hierarchical cluster analysis of differentially expressed lignin genes in different internodes of control and transgenic switchgrass.
Plant sample Wall-bound phenolics Soluble phenolics
Ester-linked Ether-linked Total phenolics Chlorogenic acid Sinapaldehyde
p-CA FA p-CA FA
Control plants
R1-I4 7.1±0.3c 4.2±0.7a 1.7±0.2c 2.7±0.5a 2.11±0.16a 0.306±0.020a 0.0040±0.0007c
R1-I3 13.8±0.3b 5.3±0.9a 3.9±0.1b 3.7±0.2a 1.53±0.03b 0.150±0.009b 0.0074±0.0011b
R1-I2 17.2±0.4ab 4.2±0.3a 5.4±0.3a 3.6±0.3a 1.56±0.07b 0.095±0.006c 0.0086±0.0007b
R1-I1 20.0±0.9a 4.4±0.9a 5.8±0.2a 3.4±0.2a 1.32±0.06c 0.075±0.003d 0.0137±0.0005a
Transgenic plants
R1-I4 4.8±0.2c 4.2±0.5a 1.0±0.1c 2.6±0.3a 3.14±0.32a 0.424±0.023a 0.0021±0.0002c
R1-I3 11.3±0.1b 5.1±0.5a 1.6±0.1b 2.6±0.3a 1.93±0.07c 0.189±0.013b 0.0049±0.0003b
R1-I2 12.5±0.2a 4.0±0.4a 1.7±0.3b 2.8±0.2a 2.34±0.03b 0.118±0.003c 0.0052±0.0002b
R1-I1 12.3±0.5a 4.4±0.9a 2.5±0.2a 2.9±0.4a 3.11±0.15a 0.129±0.019c 0.0074±0.0004a
Tab.1  Wall-bound and soluble phenolics accumulation (dry matter) in transgenic switchgrass plants (mg·g−1)
1 Rogers L A, Campbell M M C. The genetic control of lignin deposition during plant growth and development. New Phytologist, 2004, 164(1): 17–30
https://doi.org/10.1111/j.1469-8137.2004.01143.x
2 Voxeur A, Wang Y, Sibout R. Lignification: different mechanisms for a versatile polymer. Current Opinion in Plant Biology, 2015, 23(1): 83–90
https://doi.org/10.1016/j.pbi.2014.11.006 pmid: 25449731
3 Lewis N G, Yamamoto E. Lignin: occurrence, biogenesis and biodegradation. Annual Review of Plant Physiology and Plant Molecular Biology, 1990, 41(1): 455–496
https://doi.org/10.1146/annurev.pp.41.060190.002323 pmid: 11543592
4 Humphreys J M, Chapple C. Rewriting the lignin roadmap. Current Opinion in Plant Biology, 2002, 5(3): 224–229
https://doi.org/10.1016/S1369-5266(02)00257-1 pmid: 11960740
5 Bonawitz N D, Chapple C. The genetics of lignin biosynthesis: connecting genotype to phenotype. Annual Review of Genetics, 2010, 44(1): 337–363
https://doi.org/10.1146/annurev-genet-102209-163508 pmid: 20809799
6 Finkle B J, Nelson R F. Enzyme reactions with phenolic compounds: a meta-O-methyltransferase in plants. Biochimica et Biophysica Acta, 1963, 78(4): 747–749
https://doi.org/10.1016/0006-3002(63)91046-1
7 Whetten R W, MacKay J J, Sederoff R R. Recent advances in understanding lignin biosynthesis. Annual Review of Plant Physio-logy and Plant Molecular Biology, 1998, 49(49): 585–609
https://doi.org/10.1146/annurev.arplant.49.1.585 pmid: 15012247
8 Parvathi K, Chen F, Guo D, Blount J W, Dixon R A. Substrate preferences of O-methyltransferases in alfalfa suggest new pathways for 3-O-methylation of monolignols. Plant Journal, 2001, 25(2): 193–202
https://doi.org/10.1046/j.1365-313x.2001.00956.x pmid: 11169195
9 Chen L, Auh C K, Dowling P, Bell J, Lehmann D, Wang Z Y. Transgenic down-regulation of caffeic acid O-methyltransferase (COMT) led to improved digestibility in tall fescue (Festuca arundinacea). Functional Plant Biology, 2004, 31(3): 235–245
https://doi.org/10.1071/FP03254
10 Li L, Popko J L, Umezawa T, Chiang V L. 5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms. Journal of Biological Chemistry, 2000, 275(9): 6537–6545
https://doi.org/10.1074/jbc.275.9.6537 pmid: 10692459
11 Pilate G, Guiney E, Holt K, Petit-Conil M, Lapierre C, Leplé J C, Pollet B, Mila I, Webster E A, Marstorp H G, Hopkins D W, Jouanin L, Boerjan W, Schuch W, Cornu D, Halpin C. Field and pulping performances of transgenic trees with altered lignification. Nature Biotechnology, 2002, 20(6): 607–612
https://doi.org/10.1038/nbt0602-607 pmid: 12042866
12 Fu C, Mielenz J R, Xiao X, Ge Y, Hamilton C Y, Rodriguez M Jr, Chen F, Foston M, Ragauskas A, Bouton J, Dixon R A, Wang Z Y. Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(9): 3803–3808
https://doi.org/10.1073/pnas.1100310108 pmid: 21321194
13 Jouanin L, Goujon T, de Nadaï V, Martin M T, Mila I, Vallet C, Pollet B, Yoshinaga A, Chabbert B, Petit-Conil M, Lapierre C. Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity. Plant Physiology, 2000, 123(4): 1363–1374
https://doi.org/10.1104/pp.123.4.1363 pmid: 10938354
14 Piquemal J, Chamayou S, Nadaud I, Beckert M, Barrière Y, Mila I, Lapierre C, Rigau J, Puigdomenech P, Jauneau A, Digonnet C, Boudet A M, Goffner D, Pichon M. Down-regulation of caffeic acid O-methyltransferase in maize revisited using a transgenic approach. Plant Physiology, 2002, 130(4): 1675–1685
https://doi.org/10.1104/pp.012237 pmid: 12481050
15 Ralph J. Hydroxycinnamates in lignification. Phytochemistry Reviews, 2010, 9(1): 65–83
https://doi.org/10.1007/s11101-009-9141-9
16 Chen F, Srinivasa Reddy M S, Temple S, Jackson L, Shadle G, Dixon R A. Multi-site genetic modulation of monolignol biosynthesis suggests new routes for formation of syringyl lignin and wall-bound ferulic acid in alfalfa (Medicago sativa L.). Plant Journal, 2006, 48(1): 113–124
https://doi.org/10.1111/j.1365-313X.2006.02857.x pmid: 16972868
17 Palmer N A, Sattler S E, Saathoff A J, Funnell D, Pedersen J F, Sarath G. Genetic background impacts soluble and cell wall-bound aromatics in brown midrib mutants of sorghum. Planta, 2008, 229(1): 115–127
https://doi.org/10.1007/s00425-008-0814-1 pmid: 18795321
18 Bouton J H. Molecular breeding of switchgrass for use as a biofuel crop. Current Opinion in Genetics & Development, 2007, 17(6): 553–558
https://doi.org/10.1016/j.gde.2007.08.012 pmid: 17933511
19 Crowe J D, Feringa N, Pattathil S, Merritt B, Foster C, Dines D, Ong R G, Hodge D B. Identification of developmental stage and anatomical fraction contributions to cell wall recalcitrance in switchgrass. Biotechnology for Biofuels, 2017, 10(1): 184
https://doi.org/10.1186/s13068-017-0870-5 pmid: 28725264
20 Lu F, Ralph J. Detection and determination of p-coumaroylated units in lignins. Journal of Agricultural and Food Chemistry, 1999, 47(5): 1988–1992
https://doi.org/10.1021/jf981140j pmid: 10552483
21 Hatfield R D, Rancour D M, Marita J M. Grass cell walls: a story of cross-linking. Frontiers in Plant Science, 2017, 7: 2056
https://doi.org/10.3389/fpls.2016.02056 pmid: 28149301
22 Hardin C F, Fu C, Hisano H, Xiao X R, Shen H, Stewart C N, Parrott W, Dixon R A, Wang Z Y. Standardization of switchgrass sample collection for cell wall and biomass trait analysis. BioEnergy Research, 2013, 6(2): 755–762
https://doi.org/10.1007/s12155-012-9292-1
23 Xi Y, Fu C, Ge Y, Nandakumar R, Hisano H, Bouton J, Wang Z Y. Agrobacterium-mediated transformation of switchgrass and inheritance of the transgenes. BioEnergy Research, 2009, 2(4): 275–283
https://doi.org/10.1007/s12155-009-9049-7
24 Bradford M M. A dye binding assay for protein. Analytical Biochemistry, 1976, 72: 248–254
https://doi.org/10.1016/0003-2697(76)90527-3 pmid: 942051
25 Liu J, Shi R, Li Q, Sederoff R R, Chiang V L. A standard reaction condition and a single HPLC separation system are sufficient for estimation of monolignol biosynthetic pathway enzyme activities. Planta, 2012, 236(3): 879–885
https://doi.org/10.1007/s00425-012-1688-9 pmid: 22729823
26 Fu C, Sunkar R, Zhou C, Shen H, Zhang J Y, Matts J, Wolf J, Mann D G, Stewart C N Jr, Tang Y, Wang Z Y. Overexpression of miR156 in switchgrass (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production. Plant Biotechnology Journal, 2012, 10(4): 443–452
https://doi.org/10.1111/j.1467-7652.2011.00677.x pmid: 22239253
27 Shen H, Mazarei M, Hisano H, Escamilla-Trevino L, Fu C, Pu Y, Rudis M R, Tang Y, Xiao X, Jackson L, Li G, Hernandez T, Chen F, Ragauskas A J, Stewart C N Jr, Wang Z Y, Dixon R A. A genomics approach to deciphering lignin biosynthesis in switchgrass. Plant Cell, 2013, 25(11): 4342–4361
https://doi.org/10.1105/tpc.113.118828 pmid: 24285795
28 Shen H, Fu C, Xiao X, Ray T, Tang Y, Wang Z Y, Chen F. Developmental control of lignification in stems of lowland switchgrass variety Alamo and the effects on saccharification efficiency. BioEnergy Research, 2009, 2(4): 233–245
https://doi.org/10.1007/s12155-009-9058-6
29 Lapierre C, Pollet B, Rolando C. New insight into the molecular architecture of hardwood lignins by chemical degradative method. Research on Chemical Intermediates, 1995, 21(3–5): 397–412
https://doi.org/10.1007/BF03052266
30 Singleton V L, Orthofer R, Lamuela-Raventós R M. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in Enzymology, 1999, 299(2): 152–178
https://doi.org/10.1016/S0076-6879(99)99017-1
31 Fu C, Xiao X, Xi Y, Ge Y, Chen F, Bouton J, Dixon R, Wang Z Y. Downregulation of cinnamyl alcohol dehydrogenase (CAD) leads to improved saccharification efficiency in switchgrass. BioEnergy Research, 2011, 4(3): 153–164
https://doi.org/10.1007/s12155-010-9109-z
32 Nair R B, Bastress K L, Ruegger M O, Denault J W, Chapple C. The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis. Plant Cell, 2004, 16(2): 544–554
https://doi.org/10.1105/tpc.017509 pmid: 14729911
33 Goujon T, Sibout R, Pollet B, Maba B, Nussaume L, Bechtold N, Lu F, Ralph J, Mila I, Barrière Y, Lapierre C, Jouanin L. A new Arabidopsis thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters. Plant Molecular Biology, 2003, 51(6): 973–989
https://doi.org/10.1023/A:1023022825098 pmid: 12777055
34 Morreel K, Ralph J, Kim H, Lu F, Goeminne G, Ralph S, Messens E, Boerjan W. Profiling of oligolignols reveals monolignol coupling conditions in lignifying poplar xylem. Plant Physiology, 2004, 136(3): 3537–3549
https://doi.org/10.1104/pp.104.049304 pmid: 15516504
35 Rastogi S, Dwivedi U N. Down-regulation of lignin biosynthesis in transgenic Leucaena leucocephala harboring O-methyltransferase gene. Biotechnology Progress, 2006, 22(3): 609–616
https://doi.org/10.1021/bp050206+ pmid: 16739940
[1] FASE-17198-OF-WFY_suppl_1 Download
[1] Shiva O. MAKAJU, Yanqi WU, Michael P. ANDERSON, Vijaya G. KAKANI, Michael W. SMITH, Linglong LIU, Hongxu DONG, Dan CHANG. Yield-height correlation and QTL localization for plant height in two lowland switchgrass populations[J]. Front. Agr. Sci. Eng. , 2018, 5(1): 118-128.
[2] Cody F. CREECH, Blair L. WALDRON, Corey V. RAMSOM, Dale R. ZOBELL, Joseph Earl CREECH. Influence of harvest date on seed yield and quality in forage kochia[J]. Front. Agr. Sci. Eng. , 2018, 5(1): 71-79.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed