Abstract
Main conclusion
Two distinct cinnamoyl-coenzyme A reductases (CCRs) from Populus tomentosa were cloned and studied and active sites in CCRs were further identified based on sequence divergence, molecular simulation, and site-directed mutants.
Cinnamoyl-coenzyme A (CoA) reductase (CCR) is the first committed gene in the lignin-specific pathway and plays a role in the lignin biosynthesis pathway. In this study, we cloned 11 genes encoding CCR or CCR-like proteins in Populus tomentosa. An enzymatic assay of the purified recombinant P. tomentosa (Pto) CCR and PtoCCR-like proteins indicated that only PtoCCR1 and PtoCCR7 had detectable activities toward hydroxycinnamoyl-CoA esters. PtoCCR1 exhibited specificity for feruloyl-CoA, with no detectable activity for any other hydroxycinnamoyl-CoA esters. However, PtoCCR7 catalyzed p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA with a preference for feruloyl-CoA. Site-directed mutations of selected amino acids divergent between PtoCCR1 and 7, combined with modeling and docking, showed that A132 in CCR7 combined with the catalytic triad might comprise the catalytic center. In CCR7, L192, F155, and H208 were identified as the substrate-binding sites, and site-directed mutations of these amino acids showed obvious changes in catalytic efficiency with respect to both feruloyl-CoA and sinapoyl-CoA. Mutant F155Y exhibited greater catalytic efficiency for sinapoyl-CoA compared with that of wild-type PtoCCR7. Finally, recent genome duplication events provided the foundation for CCR divergence. This study further identified the active sites in CCRs and the evolutionary process of CCRs in terrestrial plants.
Similar content being viewed by others
References
Baltas M, Lapeyre C, Bedos-Belval F, Maturano M, Saint-Aguet P, Roussel L, Duran H, Grima-Pettenati J (2005) Kinetic and inhibition studies of cinnamoyl-CoA reductase 1 from Arabidopsis thaliana. Plant Physiol Biochem 43:746–753
Barakat A, Yassin NBM, Park JS, Choi A, Herr J, Carlson JE (2011) Comparative and phylogenomic analyses of cinnamoyl-CoA reductase and cinnamoyl-CoA-reductase-like gene family in land plants. Plant Sci 181:249–257
Beuerle T, Pichersky E (2002) Enzymatic synthesis and purification of aromatic coenzyme A esters. Anal Biochem 302:305–312
Bhuiyan NH, Selvaraj G, Wei Y, King J (2009) Role of lignification in plant defense. Plant Signal Behav 4:158–159
Boerjan W, Ralph J, Baucher M (2003) Lignin biosynthesis. Annu Rev Plant Biol 54:519–546
Bomati EK, Noel JP (2005) Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase. Plant Cell 17:1598–1611
Carocha V, Soler M, Hefer C, Cassan-Wang H, Fevereiro P, Myburg AA, Paiva JA, Grima-Pettenati J (2015) Genome-wide analysis of the lignin toolbox of Eucalyptus grandis. New Phytol 206:1297–1313
Chao N, Liu S-X, Liu B-M, Li N, Jiang X-N, Gai Y (2014) Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa. Planta 240:1097–1112
Consortium PGS (2011) Genome sequence and analysis of the tuber crop potato. Nature 475:189–195
Consortium TG (2012) The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485:635–641
Delano WL (2002) The PyMOL molecular graphics system (DeLano Scientific). http://www.pymol.org
Dyrløv Bendtsen J, Nielsen H, von Heijne G, Brunak S (2004) Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340:783–795
Escamilla-Treviño LL, Shen H, Uppalapati SR, Ray T, Tang Y, Hernandez T, Yin Y, Xu Y, Dixon RA (2010) Switchgrass (Panicum virgatum) possesses a divergent family of cinnamoyl CoA reductases with distinct biochemical properties. New Phytol 185:143–155
Filling C, Berndt KD, Benach J, Knapp S, Prozorovski T, Nordling E, Ladenstein R, Jörnvall H, Oppermann U (2002) Critical residues for structure and catalysis in short-chain dehydrogenases/reductases. J Biol Chem 277:25677–25684
Garcia-Mas J, Benjak A, Sanseverino W, Bourgeois M, Mir G, González VM, Hénaff E, Câmara F, Cozzuto L, Lowy E (2012) The genome of melon (Cucumis melo L.). Proc Natl Acad Sci 109:11872–11877
Goujon T, Ferret V, Mila I, Pollet B, Ruel K, Burlat V, Joseleau J-P, Barrière Y, Lapierre C, Jouanin L (2003) Down-regulation of the AtCCR1 gene in Arabidopsis thaliana: effects on phenotype, lignins and cell wall degradability. Planta 217:218–228
Guo D, Chen F, Wheeler J, Winder J, Selman S, Peterson M, Dixon RA (2001) Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases. Transgenic Res 10:457–464
Guo S, Zhang J, Sun H, Salse J, Lucas WJ, Zhang H, Zheng Y, Mao L, Ren Y, Wang Z (2013) The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions. Nat Genet 45:51–58
Hua C, Linling L, Shuiyuan C, Fuliang C, Feng X, Yan W, Dezhi J, Honghui Y, Conghua W (2014) Characterization of a cinnamoyl-CoA reductase gene in Ginkgo biloba: effects on lignification and environmental stresses. Afr J Biotechnol 11:6780–6794
Joernvall H, Persson B, Krook M, Atrian S, Gonzalez-Duarte R, Jeffery J, Ghosh D (1995) Short-chain dehydrogenases/reductases (SDR). Biochemistry 34:6003–6013
Kallberg Y, Oppermann U, Jörnvall H, Persson B (2002) Short-chain dehydrogenase/reductase (SDR) relationships: a large family with eight clusters common to human, animal, and plant genomes. Protein Sci 11:636–641
Karkonen A, Koutaniemi S (2010) Lignin biosynthesis studies in plant tissue cultures. J Integr Plant Biol 52:176–185
Kawasaki T, Koita H, Nakatsubo T, Hasegawa K, Wakabayashi K, Takahashi H, Umemura K, Umezawa T, Shimamoto K (2006) Cinnamoyl-CoA reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice. Proc Natl Acad Sci USA 103:230–235
Lacombe E, Hawkins S, Doorsselaere J, Piquemal J, Goffner D, Poeydomenge O, Boudet AM, Grima-Pettenati J (1997) Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant J 11:429–441
Lauvergeat V, Lacomme C, Lacombe E, Lasserre E, Roby D, Grima-Pettenati J (2001) Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria. Phytochemistry 57:1187–1195
Leple J-C, Dauwe R, Morreel K, Storme V, Lapierre C, Pollet B, Naumann A, Kang K-Y, Kim H, Ruel K (2007) Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. Plant Cell 19:3669–3691
Liu L, Stein A, Wittkop B, Sarvari P, Li J, Yan X, Dreyer F, Frauen M, Friedt W, Snowdon RJ (2012) A knockout mutation in the lignin biosynthesis gene CCR1 explains a major QTL for acid detergent lignin content in Brassica napus seeds. Theor Appl Genet 124:1573–1586
Lüderitz T, Grisebach H (1981) Enzymic synthesis of lignin precursors comparison of cinnamoyl-CoA reductase and cinnamyl alcohol: NADP+ dehydrogenase from spruce (Picea abies L.) and soybean (Glycine max L.). Eur j biochem 119:115–124
Lynch M, Conery JS (2000) The evolutionary fate and consequences of duplicate genes. Science 290:1151–1155
Ma Q-H (2007) Characterization of a cinnamoyl-CoA reductase that is associated with stem development in wheat. J Exp Bot 58:2011–2021
Ma Q-H, Tian B (2005) Biochemical characterization of a cinnamoyl-CoA reductase from wheat. Biol Chem 386:553–560
Moummou H, Kallberg Y, Tonfack LB, Persson B, Van der Rest B (2012) The plant short-chain dehydrogenase (SDR) superfamily: genome-wide inventory and diversification patterns. BMC Plant Biol 12:219
Mun J-H, Kwon S-J, Yang T-J, Seol Y-J, Jin M, Kim J-A, Lim M-H, Kim JS, Baek S, Choi B-S (2009) Genome-wide comparative analysis of the Brassica rapa gene space reveals genome shrinkage and differential loss of duplicated genes after whole genome triplication. Genome Biol 10:R111
Myburg AA, Grattapaglia D, Tuskan GA, Hellsten U, Hayes RD, Grimwood J, Jenkins J, Lindquist E, Tice H, Bauer D (2014) The genome of Eucalyptus grandis. Nature 510:356–362
Oppermann UC, Persson B, Filling C, Jörnvall H (1996) Structure-function relationships of SDR hydroxysteroid dehydrogenases. Enzymology and molecular biology of carbonyl metabolism 6. Springer, pp 403–415
Pan H, Zhou R, Louie GV, Mühlemann JK, Bomati EK, Bowman ME, Dudareva N, Dixon RA, Noel JP, Wang X (2014) Structural studies of cinnamoyl-CoA reductase and cinnamyl-alcohol dehydrogenase, key enzymes of monolignol biosynthesis. Plant Cell 26:3709–3727
Park S-H, Mei C, Pauly M, Ong RG, Dale BE, Sabzikar R, Fotoh H, Nguyen T, Sticklen M (2012) Downregulation of maize cinnamoyl-coenzyme a reductase via RNA interference technology causes brown midrib and improves ammonia fiber expansion-pretreated conversion into fermentable sugars for biofuels. Crop Sci 52:2687–2701
Persson B, Krook M, Jornvall H (1991) Characteristics of short-chain alcohol dehydrogenases and related enzymes. Eur J Biochem 200:537–543
Prasad NK, Vindal V, Kumar V, Kabra A, Phogat N, Kumar M (2011) Structural and docking studies of Leucaena leucocephala Cinnamoyl CoA reductase. J Mol Model 17:533–541
Raes J, Rohde A, Christensen JH, Van de Peer Y, Boerjan W (2003) Genome-wide characterization of the lignification toolbox in Arabidopsis. Plant Physiol 133:1051–1071
Ragauskas AJ, Beckham GT, Biddy MJ, Chandra R, Chen F, Davis MF, Davison BH, Dixon RA, Gilna P, Keller M (2014) Lignin valorization: improving lignin processing in the biorefinery. Science 344:1246843
Ruel K, Berrio-Sierra J, Derikvand MM, Pollet B, Thévenin J, Lapierre C, Jouanin L, Joseleau JP (2009) Impact of CCR1 silencing on the assembly of lignified secondary walls in Arabidopsis thaliana. New Phytol 184:99–113
Shi R, Sun YH, Li Q, Heber S, Sederoff R, Chiang VL (2010) Towards a systems approach for lignin biosynthesis in Populus trichocarpa: transcript abundance and specificity of the monolignol biosynthetic genes. Plant Cell Physiol 51:144–163
Sonawane P, Patel K, Vishwakarma RK, Singh S, Khan BM (2013a) In Silico mutagenesis and docking studies of active site residues suggest altered substrate specificity and possible physiological role of Cinnamoyl CoA Reductase 1 (Ll-CCRH1). Bioinformation 9:224
Sonawane P, Patel K, Vishwakarma RK, Srivastava S, Singh S, Gaikwad S, Khan BM (2013b) Probing the active site of cinnamoyl CoA reductase 1 (Ll-CCRH1) from Leucaena leucocephala. Int J Biol Macromol 60:33–38
Stöekigt J, Zenk M (1975) Chemical syntheses and properties of hydroxycinnamoyl-coenzyme A derivatives. Zeitschrift für Naturforschung C 30:352–358
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739
Tang W, Tang AY (2014) Transgenic woody plants for biofuel. J For Res 25:225–236
Tang H, Bowers JE, Wang X, Paterson AH (2010) Angiosperm genome comparisons reveal early polyploidy in the monocot lineage. Proc Natl Acad Sci 107:472–477
Tsai CJ, Harding SA, Tschaplinski TJ, Lindroth RL, Yuan Y (2006) Genome-wide analysis of the structural genes regulating defense phenylpropanoid metabolism in Populus. New Phytol 172:47–62
Tu Y, Rochfort S, Liu Z, Ran Y, Griffith M, Badenhorst P, Louie GV, Bowman ME, Smith KF, Noel JP (2010) Functional analyses of caffeic acid O-methyltransferase and cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne). Plant Cell 22:3357–3373
Tuskan GA, Difazio S, Jansson S, Bohlmann J, Grigoriev I, Hellsten U, Putnam N, Ralph S, Rombauts S, Salamov A (2006) The genome of black cottonwood, Populus trichocarpa (Torr. & Gray). Science 313:1596–1604
Van Acker R, Leplé J-C, Aerts D, Storme V, Goeminne G, Ivens B, Légée F, Lapierre C, Piens K, Van Montagu MC (2014) Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase. Proc Natl Acad Sci 111:845–850
Van de Peer Y, Maere S, Meyer A (2009) The evolutionary significance of ancient genome duplications. Nat Rev Genet 10:725–732
Van der Rest B, Danoun S, Boudet A-M, Rochange SF (2006) Down-regulation of cinnamoyl-CoA reductase in tomato (Solanum lycopersicum L.) induces dramatic changes in soluble phenolic pools. J Exp Bot 57:1399–1411
Vanholme R, Demedts B, Morreel K, Ralph J, Boerjan W (2010) Lignin biosynthesis and structure. Plant Physiol 153:895–905
Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, Bai Y, Mun J-H, Bancroft I, Cheng F (2011) The genome of the mesopolyploid crop species Brassica rapa. Nat Genet 43:1035–1039
Wang K, Wang Z, Li F, Ye W, Wang J, Song G, Yue Z, Cong L, Shang H, Zhu S (2012) The draft genome of a diploid cotton Gossypium raimondii. Nat Genet 44:1098–1103
Weng JK, Chapple C (2010) The origin and evolution of lignin biosynthesis. New Phytol 187:273–285
Weng JK, Akiyama T, Bonawitz ND, Li X, Ralph J, Chapple C (2010) Convergent evolution of syringyl lignin biosynthesis via distinct pathways in the lycophyte Selaginella and flowering plants. Plant Cell 22:1033–1045
Whetten R, Sederoff R (1995) Lignin biosynthesis. Plant Cell 7:1001–1013
Xu Q, Chen L-L, Ruan X, Chen D, Zhu A, Chen C, Bertrand D, Jiao W-B, Hao B-H, Lyon MP (2013) The draft genome of sweet orange (Citrus sinensis). Nat Genet 45:59–66
Xue J, Luo D, Xu D, Zeng M, Cui X, Li L, Huang H (2015) CCR1, an enzyme required for lignin biosynthesis in Arabidopsis, mediates cell proliferation exit for leaf development. Plant J 83:375–387
Zhao Q, Dixon RA (2014) Altering the cell wall and its impact on plant disease: from forage to bioenergy. Annu rev phytopathol 52:69–91
Zhou R, Jackson L, Shadle G, Nakashima J, Temple S, Chen F, Dixon RA (2010) Distinct cinnamoyl CoA reductases involved in parallel routes to lignin in Medicago truncatula. Proc Natl Acad Sci 107:17803–17808
Acknowledgments
This work was jointly supported by the National Natural Science Foundation [NSF 31300498 to Y.G.], High Technology Research and Development 863 Program [2011AA100203 to X.N.JIANG.], [Grant Numbers J1103516, J1310005, ITR 13047] the Basic Science Basement Facility Buildup and Talent Training Program Project from National Natural Science Foundation of China (NSFC) and PCSIRT, and the Fundamental Research Funds for the Central Universities [BLYJ201504 to N.C.].
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
425_2016_2591_MOESM1_ESM.pdf
Online Resource 1 Primers used for cloning PtoCCR and PtoCCR-like genes from P. tomentosa. Online Resource 2 Putative CCR proteins used for phylogenetic analysis. Online Resource 3 Conditions used for HPLC–MS to identify hydroxycinnamoyl-coA esters. Online Resource 4 Homology matrix of 12 sequences (DNAman). Online Resource 5 Identification of chemically synthesized hydroxycinnamoyl-CoA esters by HPLC–MS. (A–D) Chromatographs of caffeoyl-CoA, feruloyl-CoA, p-coumaroyl-CoA, and sinapoyl-CoA, respectively. (E–H) Mass spectra of caffeoyl-CoA, feruloyl-CoA, p-coumaroyl-CoA, and sinapoyl-CoA, respectively. Online Resource 6 Summary of active sites in CCRs and corresponding amino acids in 11 PtoCCR and PtoCCR-like proteins. Online Resource 7 Expression profiles of 11 PtoCCR and PtoCCR-like genes in different tissues from P. tomentosa based on microarray datasets. The transcript abundance is indicated by the blue–red gradient, as shown in the legend. Tissues or specific parts of plants are indicated. Online Resource 8 Phylogenetic trees of CCRs from core dicots. (A) Phylogenetic tree of CCRs from eurosids II. (B) Phylogenetic tree of CCRs from eurosids I. AtrCCR was used as the root. Recent whole genome duplications are marked with blue-filled ellipses (PDF 570 kb)
Rights and permissions
About this article
Cite this article
Chao, N., Li, N., Qi, Q. et al. Characterization of the cinnamoyl-CoA reductase (CCR) gene family in Populus tomentosa reveals the enzymatic active sites and evolution of CCR. Planta 245, 61–75 (2017). https://doi.org/10.1007/s00425-016-2591-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00425-016-2591-6