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

Abstract

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.

Keywords

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

Abbreviations

cDNA

Complementary DNA

EDTA

Ethylene diamine tetraacetie acid

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

IPTG

Isopropyl β-d-1-thiogalactopyranoside

kDa

Kilodaltons

MIO

3,5-Dihydro-5-methylidine-4H-imidazol-4-one

ORF

Open reading frame

PAL

Phenylalanine ammonia-lyase

PBS

Phosphate-buffered saline

RT-PCR

Reverse transcription-polymerase chain reaction

RACE

Rapid amplification of cDNA ends

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TAL

Tyrosine ammonia-lyase

UTR

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)

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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

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