Plant Cell Reports

, Volume 32, Issue 12, pp 1827–1841 | Cite as

Wood chemistry analysis and expression profiling of a poplar clone expressing a tyrosine-rich peptide

  • Yi Xu
  • Chin-Fu Chen
  • Tina P. Thomas
  • Parastoo Azadi
  • Brett Diehl
  • Chung-Jui Tsai
  • Nicole Brown
  • John E. Carlson
  • Ming Tien
  • Haiying LiangEmail author
Original Paper


Key message

Our study has identified pathways and gene candidates that may be associated with the greater flexibility and digestibility of the poplar cell walls.


With the goal of facilitating lignin removal during the utilization of woody biomass as a biofuel feedstock, we previously transformed a hybrid poplar clone with a partial cDNA sequence encoding a tyrosine- and hydroxyproline-rich glycoprotein from parsley. A number of the transgenic lines released more polysaccharides following protease digestion and were more flexible than wild-type plants, but otherwise normal in phenotype. Here, we report that overexpression of the tyrosine-rich peptide encoding sequence in these transgenic poplar plants did not significantly alter total lignin quantity or quality (S/G lignin ratio), five- and six-carbon sugar contents, growth rate, or susceptibility to a major poplar fungal pathogen, Septoria musiva. Whole-genome microarray analysis revealed a total of 411 differentially expressed transcripts in transgenic lines, all with decreased transcript abundance relative to wild-type plants. Their corresponding genes were overrepresented in functional categories such as secondary metabolism, amino acid metabolism, and energy metabolism. Transcript abundance was decreased primarily for five types of genes encoding proteins involved in cell-wall organization and in lignin biosynthesis. The expression of a subset of 19 of the differentially regulated genes by qRT-PCR validated the microarray results. Our study has identified pathways and gene candidates that may be the underlying cause for the enhanced flexibility and digestibility of the stems of poplar plants expressing the TYR transgene.


Cell-wall composition Differential gene expression Poplar Pyrolysis molecular beam mass spectrometry (pyMBMS) Tyrosine-rich peptide 



The authors thank Mark Davis and Rob Sykes at the National Renewable Energy Laboratory (NREL) and Gary F. Peter and Jianxing Zhang at University of Florida for their assistance in the pyMBMS analysis, and Abdelali Barakat for his advice in primer design for the qRT-PCR work. This study was supported by a US Department of Energy’s Biosciences Program grant (DF-FG02-07ER) to Ming Tien, a National Institute of Food and Agriculture/USDA grant to Haiying Liang (project number SC-1700324, technical contribution No. 5923 of the Clemson University Experiment Station), and the World Class University Project R31-2009-000-20025-0 grant by the Ministry of Education, Science and Technology of Korea to John E. Carlson. The pyMBMS facility at the University of Georgia was supported by a DOE grant DE-EE-0000410.

Supplementary material

299_2013_1496_MOESM1_ESM.doc (141 kb)
Supplementary material 1 (DOC 141 kb)
299_2013_1496_MOESM2_ESM.xlsx (386 kb)
Supplementary material 2 (XLSX 386 kb)
299_2013_1496_MOESM3_ESM.xlsx (14 kb)
Supplementary material 3 (XLSX 13 kb)


  1. Abramoff MD, Magalhaes PJ, Ram SJ (2004) Image Processing with ImageJ. Biophotonics Int 11(7):36–42Google Scholar
  2. Aniszewski T, Lieberei R, Gulewicz K (2008) Research on catecholases, laccases and cresolases in plants. Recent progress and future needs. Acta Biol Cracov Bot 50:7–18Google Scholar
  3. Azar C (2003) Emerging scarcities: bioenergy-food competition in a carbon constrained world. In: Simpson D, Toman M, Ayres R (eds) Scarcity and growth in the new millennium. Resources for the future. Johns Hopkins University Press, BaltimoreGoogle Scholar
  4. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. JR Stat Soc Ser B (Methodol) 57:289–300Google Scholar
  5. Berthet S, Demont-Caulet N, Pollet B, Bidzinski P, Cezard L, P Le Bris, Borrega N, Herve J, Blondet E, Balzergue S, Lapierre C, Jouanin L (2011) Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems. Plant Cell 23:1124–1137PubMedCrossRefGoogle Scholar
  6. Chang S, Puryear J, Cairney J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116CrossRefGoogle Scholar
  7. Churchill GA (2002) Fundamentals of experimental design for cDNA microarrays. Nat Genet 32(Suppl):490–495PubMedCrossRefGoogle Scholar
  8. Dauwe R, Morreel K, Goeminne G, Gielen B, Rohde A, Van Beeumen J, Ralph J, Boudet AM, Kopka J, Rochange SF, Halpin C, Messens E, Boerjan W (2007) Molecular phenotyping of lignin-modified tobacco reveals associated changes in cell-wall metabolism, primary metabolism, stress metabolism and photorespiration. Plant J 52:263–285PubMedCrossRefGoogle Scholar
  9. Deepak S, Shailasree S, Kini RK, Hause B, Shetty SH, Mithöfer A (2007) Role of hydroxyproline-rich glycoproteins in resistance of pearl millet against downy mildew pathogen Sclerospora graminicola. Planta 226:323–333PubMedCrossRefGoogle Scholar
  10. Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA (2003) DAVID: database for annotation, visualization, and integrated discovery. Genome Biol 4:P3PubMedCrossRefGoogle Scholar
  11. Dharmawardhana DP, Ellis BE, Carlson JE (1999) cDNA cloning and heterologous expression of coniferin β-glucosidase. Plant Mol Biol 40:365–372PubMedCrossRefGoogle Scholar
  12. Evans RJ, Milne TA (1987) Molecular characterization of the pyrolysis of biomass. Energy Fuels 1:123–137CrossRefGoogle Scholar
  13. Gallego-Giraldo L, Jikumaru Y, Kamiya Y, Tang Y, Dixon RA (2011) Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.). New Phytol 190:627–639PubMedCrossRefGoogle Scholar
  14. Gray KA, Zhao L, Emptage M (2006) Bioethanol. Curr Opin Chem Biol 10:141–146PubMedCrossRefGoogle Scholar
  15. Gray-Mitsumune M, Molitor EK, Cukovic D, Carlson JE, Douglas CJ (1999) Developmentally regulated patterns of expression directed by poplar PAL promoters in transgenic tobacco and poplar. Plant Mol Biol 39:657–669PubMedCrossRefGoogle Scholar
  16. Grima-Pettenati J, Soler M, Camatgo ELO, Wang H (2012) Transcriptional regulation of the lignin biosynthetic pathway revisited: new players and insights. Lignins Biosynth Biodegrad Bioeng 61:173–218Google Scholar
  17. Hu WJ, Harding SA, Lung J, Popko JL, Ralph J, Stokke DD, Tsai C-J, Chiang VL (1999) Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat Biotechnol 17:808–812PubMedCrossRefGoogle Scholar
  18. Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4:44–57CrossRefGoogle Scholar
  19. Jackson LA, Shadle GL, Zhou R, Nakashima J, Chen F, Dixon RA (2008) Improving saccharification efficiency of alfalfa stems through modification of the terminal stages of monolignol biosynthesis. Bioenerg Res 1:180–192CrossRefGoogle Scholar
  20. John RP, Anisha GS, Nampoothiri KM, Pandey A (2011) Micro and macroalgal biomass: a renewable source for bioethanol. Biores Technol 102:186–193CrossRefGoogle Scholar
  21. Jouanin L, Goujon T, de Nadai V, Martin MT, Mila I, Vallet C, Pollet B, Yoshinaga A, Chabbert B, Petit-Conil M, Lapierre C (2000) Lignification in transgenic poplars with extremely reduced caffeic acid O-methyltransferase activity. Plant Physiol 123:1363–1373PubMedCrossRefGoogle Scholar
  22. Kawalleck P, Schmelzer E, Hahlbrock K, Somssich IE (1995) Two pathogen-responsive genes in parsley encode a tyrosine-rich hydroxyproline-rich glycoprotein (HRGP) and an anionic peroxidase. Mol Gen Genet 247:444–452PubMedCrossRefGoogle Scholar
  23. 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. PNAS 103:230–235PubMedCrossRefGoogle Scholar
  24. Krupinsky JM (1989) Variability in Septoria musiva in aggressiveness. Ecol Epidemiol 79:413–416Google Scholar
  25. Leple JC, Dauwe R, Morreel K, Storme V, Lapierre C, Pollet B, Naumann A, Kang KY, Kim H, Ruel K, Lefèbvre A, Joseleau JP, Grima-Pettenati J, De Rycke R, Andersson-Gunnerås S, Erban A, Fehrle I, Petit-Conil M, Kopka J, Polle A, Messens E, Sundberg B, Mansfield SD, Ralph J, Pilate G, Boerjan W (2007) Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure. Plant Cell 19:3669–3691PubMedCrossRefGoogle Scholar
  26. Li L, Popko J, Umezawa T, Chiang V (2000) 5-Hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms. J Bio Chem 275:6537–6545CrossRefGoogle Scholar
  27. Li X, Bonawitz ND, Weng JK, Chapple C (2010) The growth reduction associated with repressed lignin biosynthesis in Arabidopsis thaliana is independent of flavonoids. Plant Cell 22:1620–1632PubMedCrossRefGoogle Scholar
  28. Liang H, Maynard CA, Allen RD, Powell WA (2001) Increased Septoria Musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene. Plant Mol Biol 45:619–629PubMedCrossRefGoogle Scholar
  29. Liang M, Davis E, Gardner D, Cai X, Wu Y (2006) Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis. Planta 224:1185–1196PubMedCrossRefGoogle Scholar
  30. Liang H, Frost CJ, Wei X, Brown NR, Carlson JE, Tien M (2008) Improved sugar release from lignocellulosic material by introducing a tyrosine-rich cell wall peptide gene in poplar. Clean Soil Air Water 36:662–668CrossRefGoogle Scholar
  31. Lin SY, Dence CW (eds) (1992) Methods in lignin chemistry. Springer, Berlin Google Scholar
  32. Mayer AM, Staples RC (2002) Laccase: new functions for an old enzyme. Phytochemistry 60:551–565PubMedCrossRefGoogle Scholar
  33. Morey JS, Ryan JC, Van Dolah FM (2006) Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol Proced Online 8:175–193PubMedCrossRefGoogle Scholar
  34. Novaes E, Osorio L, Drost DR, Miles BL, Boaventura-Novaes CRD, Benedict C, Dervinis C, Yu Q, Sykes R, Davis M, Martin TA, Peter GF, Kirst M (2009) Quantitative genetic analysis of biomass and wood chemistry of Populus under different nitrogen levels. New Phytol 182:878–890PubMedCrossRefGoogle Scholar
  35. Pedersen JF, Vogel KP, Funnell DL (2005) Impact of reduced lignin on plant fitness. Crop Sci 45:812–819CrossRefGoogle Scholar
  36. Prashant S, Srilakshmi Sunita M, Pramod S, Gupta RK, Anil Kumar S, Rao Karumanchi S, Rawal SK, Kavi Kishor PB (2011) Down-regulation of Leucaena leucocephala cinnamoyl CoA reductase (LlCCR) gene induces significant changes in phenotype, soluble phenolic pools and lignin in transgenic tobacco. Plant Cell Rep 30:2215–2231PubMedCrossRefGoogle Scholar
  37. Quackenbush J (2002) Microarray data normalization and transformation. Nat Genet 32:496–501PubMedCrossRefGoogle Scholar
  38. Ragauskas AJ, Williams CK, Davison BH, Briovsek G, Cairney J, Eckert CA, Frederick WJ, Hallett JP, Leak DJ, Liotta CL, Mielenz JR, Murphy R, Templer R, Tschaplinski T (2006) The path forward for biofuels and biomaterials. Science 311:484–489PubMedCrossRefGoogle Scholar
  39. Ralph J, Lapierre C, Lu FC, Marita JM, Pilate G, Van Doorsselaere J, Boerjan W, Jouanin L (2001) NMR evidence for benzodioxane structures resulting from incorporation of 5-hydroxyconiferyl alcohol into lignins of O-methyltransferase-deficient poplars. J Agric Food Chem 49:86–91PubMedCrossRefGoogle Scholar
  40. Ranocha P, McDougall G, Hawkins S, Sterjiades R, Borderies G, Stewart D, Cabanes-Macheteau M, Boudet AM, Goffner D (1999) Biochemical characterization, molecular cloning and expression of laccases—a divergent gene family—in poplar. Eur J Biochem 259:485–495PubMedCrossRefGoogle Scholar
  41. Ranocha P, Chabannes M, Chamayou S, Danoun S, Jauneau A, Boudet AM, Goffner D (2002) Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar. Plant Physiol 129:145–155PubMedCrossRefGoogle Scholar
  42. Rathmann R, Szklo A, Schaeffer R (2010) Land use competition for production of food and liquid biofuels: an analysis of the arguments in the current debates. Renew Energy 35:14–22CrossRefGoogle Scholar
  43. Rogers LA, Campbell MM (2004) The genetic control of lignin deposition during plant growth and development. New Phytol 164:17–30CrossRefGoogle Scholar
  44. Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386Google Scholar
  45. Sanchez OJ, Cardona CA (2008) Trends in biotechnological production of fuel ethanol from different feedstocks. Biores Technol 99:5270–5295CrossRefGoogle Scholar
  46. Shadle G, Chen F, Srinivasa Reddy MS, Jackson L, Nakashima J, Dixon RA (2007) Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase in transgenic alfalfa affects lignification, development and forage quality. Phytochemistry 68:1530–1536CrossRefGoogle Scholar
  47. Shailasreea S, Kini RK, Deepaka S, Kumudinia BS, Shetty HS (2004) Accumulation of hydroxyproline-rich glycoproteins in pearl millet seedlings in response to Sclerospora graminicola infection. Plant Sci 167:1227–1234CrossRefGoogle Scholar
  48. Shi R, Sun Y-H, 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 51:144–163PubMedCrossRefGoogle Scholar
  49. Showalter AM, Bell JN, Cramer CL, Bailey JA, Varner JE, Lamb CJ (1985) Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection. Proc Natl Acad Sci USA 82:6551–6555PubMedCrossRefGoogle Scholar
  50. Simmons BA, Loque D, Ralph J (2010) Advances in modifying lignin for enhanced biofuel production. Curr Opin Plant Biol 13:312–319CrossRefGoogle Scholar
  51. Sokal RR, Rohlf FJ (1995) Biometry, 3rd edn. W. H. Freeman and Co, New YorkGoogle Scholar
  52. Sticklen MB (2008) Plant genetic engineering to improve biomass characteristics for biofuels. Curr Opin Biotechnol 9:433–443Google Scholar
  53. Storey JD, Tibshirani R (2003) Statistical significance for genomewide studies. Proc Natl Acad Sci USA 100:9440–9445PubMedCrossRefGoogle Scholar
  54. Sykes R, Kodrzycki B, Tuskan G, Foutz K, Davis M (2008) Within tree variability of lignin composition in Populus. Wood Sci Technol 42:649–661CrossRefGoogle Scholar
  55. Tamasloukht B, Lam MWQ, Martinez Y, Tozo K, Barbier O, Jourda C, Jauneau A, Borderies G, Balzergue S, Renou JP, Huguet S, Martinant JP, Tatout C, Lapierre C, Barriere Y, Goffner D, Pichon M (2011) Characterization of a cinnamoyl-CoA reductase 1 (CCR1) mutant in maize: effects on lignification, fibre development, and global gene expression. J Exp Bot 62:3837–3848PubMedCrossRefGoogle Scholar
  56. Thurston CF (1994) The structure and function of fungal laccases. Microbiology 140:19–26CrossRefGoogle Scholar
  57. Tronchet M, Balagué C, Kroj T, Jouanin Roby D (2010) Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis. Mol Plant Pathol 11:83–92PubMedCrossRefGoogle Scholar
  58. Tsai C-J, Popko JL, Mielke MR, Hu WJ, Podila GK, Chiang VL (1998) Suppression of O-methyltransferase gene by homologous sense transgene in quaking aspen causes red-brown wood phenotypes. Plant Physiol 117:101–112PubMedCrossRefGoogle Scholar
  59. Tsai C-J, Ranjan P, DiFazio SP, Tuskan GA, Johnson V (2011) Poplar genome microarrays. In: Joshi CP, DiFazio SP, Kole C (eds) Genetics, genomics and breeding of Poplar. Science Publishers, Enfield, New Hampshire, pp 112–127Google Scholar
  60. Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:5116–5121PubMedCrossRefGoogle Scholar
  61. van der Rest B, Danoun S, Boudet AM, Rochange SF (2006) Down-regulation of cinnamoyl-CoA reductase in tomato (Solanum lycopersicum L.) induces dramatic changes in soluble phenolic pools. J Exp Bot 57:1399–1411PubMedCrossRefGoogle Scholar
  62. Van Doorsselaere J, Baucher M, Chognot E, Chabbert B, Tollier M-T, Petit-Conil M, Leplé J-C, Pilate G, Cornu D, Monties B, Van Montagu M, Inzé D, Boerjan W, Jouanin L (1995) A novel lignin in poplar trees with a reduced caffeic acid 5-hydroxyferulic acid O-methyltransferase activity. Plant J 8:855–864CrossRefGoogle Scholar
  63. Voelker SL, Lachenbruch B, Meinzer FC, Jourdes M, Ki C, Patten AM, Davin LB, Lewis NG, Tuskan GA, Gunter L, Decker SR, Selig MJ, Sykes R, Himmel ME, Kitin P, Shevchenko O, Strauss SH (2010) Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar. Plant Physiol 154:874–886PubMedCrossRefGoogle Scholar
  64. Wagner A, Ralph J, Akiyama T, Flint H, Phillips L, Torr K, Nanayakkara B, Te Kiri L (2007) Exploring lignification in conifers by silencing hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase in Pinus radiata. Proc Natl Acad Sci USA 104:11856–11861PubMedCrossRefGoogle Scholar
  65. Wang J, Wang C, Zhu M, Yu Y, Zhang Y, Wei Z (2008) Generation and characterization of transgenic poplar plants overexpressing a cotton laccase gene. Plant Cell Tissue Organ Cult 93:303–310CrossRefGoogle Scholar
  66. Weng JK, Li X, Bonawitz ND, Chapple C (2008) Emerging strategies of lignin engineering and degradation for cellulosic biofuel production. Curr Opin Biotechnol 19:166–172PubMedCrossRefGoogle Scholar
  67. Yang YH, Speed T (2002) Design issues for cDNA microarray experiments. Nat Rev Genet 3:579–588PubMedGoogle Scholar
  68. Yoshida H (1883) Chemistry of lacquer (urushi). J Chem Soc 43:472–486CrossRefGoogle Scholar
  69. Zhong R, Lee C, Zhou J, McCarthy RL, Ye Z-H (2008) A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell 20:2763–2782PubMedCrossRefGoogle Scholar
  70. Zhou J, Lee C, Zhong R, Ye ZH (2009) MYB58 and MYB63 are transcriptional activators of the lignin biosynthetic pathway during secondary cell wall formation in Arabidopsis. Plant Cell 21:248–266PubMedCrossRefGoogle Scholar
  71. 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 USA 107:17803–17808PubMedCrossRefGoogle Scholar
  72. Zhu J, Pan X (2010) Woody biomass pretreatment for cellulosic ethanol production: technology and energy consumption evaluation. Biores Technol 101(13):4992–5002CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Yi Xu
    • 1
  • Chin-Fu Chen
    • 2
  • Tina P. Thomas
    • 3
  • Parastoo Azadi
    • 4
  • Brett Diehl
    • 5
  • Chung-Jui Tsai
    • 6
  • Nicole Brown
    • 5
  • John E. Carlson
    • 7
    • 8
  • Ming Tien
    • 9
  • Haiying Liang
    • 1
    Email author
  1. 1.Department of Genetics and BiochemistryClemson UniversityClemsonUSA
  2. 2.Center for Molecular StudiesGreenwood Genetic CenterGreenwoodUSA
  3. 3.Bioenergy Systems Research InstituteUniversity of GeorgiaAthensUSA
  4. 4.Complex Carbohydrate Research CenterUniversity of GeorgiaAthensUSA
  5. 5.Department of Agricultural and Biological EngineeringPennsylvania State UniversityUniversity ParkUSA
  6. 6.Warnell School of Forestry and Natural Resources and Department of GeneticsUniversity of GeorgiaAthensUSA
  7. 7.The Department of Ecosystem Science and Management and The Department of Plant SciencePennsylvania State UniversityUniversity ParkUSA
  8. 8.Department of Bioenergy Science and Technology (World Class University)Chonnam National University, Buk-GuGwangjuKorea
  9. 9.Department of Biochemistry and Molecular BiologyPennsylvania State UniversityUniversity ParkUSA

Personalised recommendations