Plant Cell Reports

, Volume 31, Issue 7, pp 1345–1356 | Cite as

Characterization and primary functional analysis of phenylalanine ammonia-lyase gene from Phyllostachys edulis

Original Paper


Phenylalanine ammonia-lyase (PAL) catalyzes the first reaction in phenylpropanoid pathway leading to the production of phenolic compounds with a wide range of biological functions. The cDNA encoding PAL was isolated from Phyllostachys edulis by reverse transcription-polymerase chain reaction (RT-PCR) and by 5′ and 3′ rapid amplification of cDNA ends, and was designated as PePAL. The full length of PePAL was 2,503 bp which contained an open reading frame (ORF) encoding a peptide of 701 amino acids, with a theoretic isoelectric point of 6.49 and a calculated molecular mass of 75.7 kDa. PePAL was heterologously expressed in Escherichia coli and the recombinant proteins exhibited both PAL and tyrosine ammonia-lyase (TAL) activities. The optimum temperature and pH of the recombinant PePAL were 50 °C and 8.5–9.0, respectively. The Km and Vmax values for l-phenylalanine was calculated as 422 μM and 45.9 nM s−1, while for l-tyrosine were 78 μM and 7.09 nM s−1, respectively. Tissue-specific expression assay showed that PePAL expressed highest in stem and sheath, followed by leaf, and lowest in root. Though the accumulation of PePAL proteins was observed in all these four organs by Western blotting, the highest was detected in leaf. Immunohistochemistry study showed that PePAL was localized primarily in vascular bundles and part of sclerenchyma cells of leaf, sheath and root. These results suggested that PePAL had similar expression pattern and biochemical properties with PALs in other plants, which laid the basis for molecular engineering to improve the quality of bamboo products.

Key message PePAL was a protein with bifunctional enzyme activities of PAL and TAL as shown in vitro assays, and localized primarily in bamboo vascular bundles.


Phyllostachys edulis Phenylalanine ammonia-lyase Expression analysis Enzymatic assay in vitro Immunohistochemistry 



Complementary DNA


Ethylene diamine tetraacetie acid


Glyceraldehyde-3-phosphate dehydrogenase


Isopropyl β-d-1-thiogalactopyranoside






Open reading frame


Phenylalanine ammonia-lyase


Phosphate-buffered saline


Reverse transcription-polymerase chain reaction


Rapid amplification of cDNA ends


Sodium dodecyl sulfate polyacrylamide gel electrophoresis


Tyrosine ammonia-lyase


Untranslated regions

Supplementary material

299_2012_1253_MOESM1_ESM.doc (48 kb)
Supplementary material 1 (DOC 48 kb)
299_2012_1253_MOESM2_ESM.doc (118 kb)
Supplementary material 2 (DOC 118 kb)


  1. Anterola AM, Jeon JH, Davin LB, Lewis NG (2002) Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism. J Biol Chem 277:18272–18280PubMedCrossRefGoogle Scholar
  2. Berüter J, Feusi MES (1997) The effect of girdling on carbohydrate partitioning in the growing apple fruit. J Plant Physiol 151:277–285CrossRefGoogle Scholar
  3. Bolwell GP (1992) A role for phosphorylation in the down-regulation of phenylalanine ammonia-lyase in suspension-cultured cells of French bean. Phytochemistry 31:4081–4086CrossRefGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. Chaman ME, Copaja SV, Argandona VH (2003) Relationships between salicylic acid content, phenylalanine ammonia-lyase (PAL) activity, and resistance of barley to aphid infestation. J Agric Food Chem 51:2227–2231PubMedCrossRefGoogle Scholar
  6. Cochrane FC, Davin LB, Lewis NG (2004) The Arabidopsis phenylalanine ammonia-lyase gene family: kinetic characterization of the four PAL isoforms. Phytochemistry 65:1557–1564PubMedCrossRefGoogle Scholar
  7. Dixon RA, Paiva NL (1995) Stress induced phenylpropanoid metabolism. Plant Cell 7:1085–1097PubMedGoogle Scholar
  8. Gui Y, Wang S, Quan L, Zhou C, Long S, Zheng H, Jin L, Zhang X, Ma N, Fan L (2007) Genome size and sequence composition of moso bamboo: a comparative study. Sci China C Life Sci 50:700–705PubMedCrossRefGoogle Scholar
  9. Guo J, Wang WH (2009) Characterization of the phenylalanine ammonia-lyase gene (SlPAL5) from tomato (Solanum lycopersicum L.). Mol Biol Rep 36:1579–1585PubMedCrossRefGoogle Scholar
  10. Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenyl-propanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40:347–369CrossRefGoogle Scholar
  11. Hattori T, Nishiyawa A, Shimada M (1999) Induction of l-phenylalanine ammonia-lyase and suppression of veratryl alcohol biosynthesis by exogenously added l-phenylalanine in a whiterot fungus Phanerochaete chrysosporium. FEMS Microbiol Lett 179:305–309PubMedGoogle Scholar
  12. Havir EA, Reid PD, Marsh HVJ (1971) l-phenylalanine ammonia-lyase (maize). Plant Physiol 48:130–136PubMedCrossRefGoogle Scholar
  13. Hsieh LS, Ma GJ, Yang CC, Lee PD (2010a) Cloning, expression, site-directed mutagenesis and immunolocalization of phenylalanine ammonia-lyase in Bambusa oldhamii. Phytochemistry 71:1999–2009PubMedCrossRefGoogle Scholar
  14. Hsieh LS, Yeh CS, Pan HC, Cheng CY, Yang CC, Lee PD (2010b) Cloning and expression of a phenylalanine ammonia-lyase gene (BoPAL2) from Bambusa oldhamii in Escherichia coli and Pichia pastoris. Pro Exp Purif 71:224–230CrossRefGoogle Scholar
  15. Hsieh LS, Hsieh YL, Yeh CS, Cheng CY, Yang CC, Lee PD (2011) Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from B. oldhamii. Mol Biol Rep 38:283–290PubMedCrossRefGoogle Scholar
  16. Huazhong agriculture university plant microtechnique group (1984) The whole-mount staining with Ehrilich’s hematoxylin and the double drop staining with safranin-fast green on paraffin method. Chinese bulletin of botany 3(6):56–59Google Scholar
  17. Jiang ZH (2002) Bamboo and rattan in the world. LiaoNing Science and Technology Published House Press, ShenyangGoogle Scholar
  18. Jones DH (1984) Phenylalanine ammonia-lyase: regulation of its induction, and its role in plant development. Phytochemistry 23:1349–1359CrossRefGoogle Scholar
  19. Joos HJ, Hahlbrock K (1992) Phenylalanine ammonia-lyase in potato (Solanum tuberosum L.) Genomic complexity, structural comparison of two selected genes and modes of expression. Eur J Biochem 204:621–629PubMedCrossRefGoogle Scholar
  20. Kervinen T, Peltonen S, Utriainen M, Kangasjärvi J, Teeri TH, Karjalainen R (1997) Cloning and characterization of cDNA clones encoding phenylalanine ammonia-lyase in barley. Plant Sci 123:143–150CrossRefGoogle Scholar
  21. Kim SH, Kronstad JW, Ellis BE (1996) Purification and characterization of phenylalanine ammonia-lyase from Ustilago maydis. Phytochemistry 43:351–357CrossRefGoogle Scholar
  22. Kyndt JA, Meyer TE, Cusanovich MA, Van Beeumen JJ (2002) Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein. FEBS Lett 512:240–244PubMedCrossRefGoogle Scholar
  23. Liang X, Dron M, Cramer CL, Dixon RA, Lamb CJ (1989) Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues. J Biol Chem 264:14486–14492PubMedGoogle Scholar
  24. Lim HW, Park SS, Lim CJ (1997) Purification and properties of phenylalanine ammonia-lyase from leaf mustard. Mol Cells 7:715–720PubMedGoogle Scholar
  25. Lois R, Dietrich A, Hahlbrock K, Schulz W (1989) A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identification of elicitor and light responsive cis-acting elements. EMBO J 8:1641–1648PubMedGoogle Scholar
  26. Lu BB, Du Z, Ding RX, Zhang L, Yu XJ, Liu CH, Chen WS (2006) Cloning and characterization of a differentially expressed phenylalanine ammonia-lyase gene (IiPAL) after genome duplication from tetraploid Isatis indigotica Fort. J Integr Plant Biol 48:1439–1449CrossRefGoogle Scholar
  27. MacDonald MJ, D’Cunha GB (2007) A modern view of phenylalanine ammonialyase. Biochem Cell Biol 85:273–282PubMedCrossRefGoogle Scholar
  28. Moffitt MC, Louie GV, Bowman ME, Pence J, Noel JP, Moore BS (2007) Discovery of the cyanobacterial phenylalanine ammonia-lyases: kinetic and structural characterization. Biochemistry 46:1004–1012PubMedCrossRefGoogle Scholar
  29. Nakashima J, Mizuno T, Takabe K, Fujita M, Saiki H (1997) Direct visualization of lignifying secondary wall thickenings in Zinnia elegans cells in culture. Plant Cell Physiol 38:818–827CrossRefGoogle Scholar
  30. Nugroho LH, Verberne MC, Verpoorte R (2002) Activities of enzymes involved in the phenylpropanoid pathway in constitutively salicylic acid-producing tobacco plants. Plant Physiol Biochem 40:755–760CrossRefGoogle Scholar
  31. Ohl S, Hedrick SA, Choy J, Lamb CJ (1990) Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis. Plant Cell 2:837–848PubMedGoogle Scholar
  32. Okada T, Mikage M, Sekita S (2008) Molecular characterization of the phenylalanine ammonia-lyase from Ephedra sinica. Biol Pharm Bull 12:2194–2199CrossRefGoogle Scholar
  33. Peng Z, Lu T, Li L, Liu X, Gao Z, Hu T, Yang X, Feng Q, Guan J, Weng Q, Fan D, Han B, Jiang Z (2010) Genome-wide characterization of the biggest grass, bamboo, based on 10,608 putative full-length cDNA sequences. BMC Plant Bio 10:116CrossRefGoogle Scholar
  34. Reddy JT, Korth KL, Wesley SV, Howles PA, Rasmussen S, Lamb C, Dixon RA (2000) Post-transcriptional regulation of phenylalanine ammonia-lyase expression in tobacco following recovery from gene silencing. Biol Chem 8:655–665CrossRefGoogle Scholar
  35. Rétey J (2003) Discovery and role of methylidene imidazolone, a highly electrophilic prosthetic group. Biochim Biophys Acta 1647:179–184PubMedGoogle Scholar
  36. Rösler J, Krekel F, Amrhein N, Schmid J (1997) Maize phenylalanine ammonialyase has tyrosine ammonia-lyase activity. Plant Physiol 113:175–179PubMedCrossRefGoogle Scholar
  37. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning—a laboratory manual, 2nd edn. Cold Spring Harbour Lab Press, New YorkGoogle Scholar
  38. Sarma AD, Sharma R (1999) Purification and characterization of UV-B induced phenylalanine ammonia-lyase from rice seedling. Phytochemistry 50:729–737CrossRefGoogle Scholar
  39. Song J, Wang Z (2009) Molecular cloning, expression and characterization of a phenylalanine ammonia-lyase gene (SmPAL1) from Salvia miltiorrhiza. Mol Biol Rep 36:939–952PubMedCrossRefGoogle Scholar
  40. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599PubMedCrossRefGoogle Scholar
  41. Wanner LA, Li G, Ware D, Somssich IE, Davis KR (1995) The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana. Plant Mol Biol 27:327–338PubMedCrossRefGoogle Scholar
  42. Xu F, Cai R, Cheng S, Du H, Wang Y, Cheng S (2008) Molecular cloning, characterization and expression of phenylalanine ammonia-lyase gene from Ginkgo biloba. Afr J Biotechnol 7:721–729Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  1. 1.State Forestry Administration Key Open Laboratory on Bamboo and Rattan Science and TechnologyInternational Center for Bamboo and RattanBeijingPeople’s Republic of China
  2. 2.Research Institute of ForestryChinese Academy of ForestryBeijingPeople’s Republic of China

Personalised recommendations