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Planta

, Volume 240, Issue 5, pp 1097–1112 | Cite as

Molecular cloning and functional analysis of nine cinnamyl alcohol dehydrogenase family members in Populus tomentosa

  • Nan Chao
  • Shu-Xin Liu
  • Bing-Mei Liu
  • Ning Li
  • Xiang-Ning Jiang
  • Ying GaiEmail author
Original Article

Abstract

Main conclusion

Nine CAD/CAD-like genes in P. tomentosa were classified into four classes based on expression patterns, phylogenetic analysis and biochemical properties with modification for the previous claim of SAD.

Cinnamyl alcohol dehydrogenase (CAD) functions in monolignol biosynthesis and plays a critical role in wood development and defense. In this study, we isolated and cloned nine CAD/CAD-like genes in the Populus tomentosa genome. We investigated differential expression using microarray chips and found that PtoCAD1 was highly expressed in bud, root and vascular tissues (xylem and phloem) with the greatest expression in the root. Differential expression in tissues was demonstrated for PtoCAD3, PtoCAD6 and PtoCAD9. Biochemical analysis of purified PtoCADs in vitro indicated PtoCAD1, PtoCAD2 and PtoCAD8 had detectable activity against both coniferaldehyde and sinapaldehyde. PtoCAD1 used both substrates with high efficiency. PtoCAD2 showed no specific requirement for sinapaldehyde in spite of its high identity with so-called PtrSAD (sinapyl alcohol dehydrogenase). In addition, the enzymatic activity of PtoCAD1 and PtoCAD2 was affected by temperature. We classified these nine CAD/CAD-like genes into four classes: class I included PtoCAD1, which was a bone fide CAD with the highest activity; class II included PtoCAD2, -5, -7, -8, which might function in monolignol biosynthesis and defense; class III genes included PtoCAD3, -6, -9, which have a distinct expression pattern; class IV included PtoCAD12, which has a distinct structure. These data suggest divergence of the PtoCADs and its homologs, related to their functions. We propose genes in class II are a subset of CAD genes that evolved before angiosperms appeared. These results suggest CAD/CAD-like genes in classes I and II play a role in monolignol biosynthesis and contribute to our knowledge of lignin biosynthesis in P. tomentosa.

Keywords

Cinnamyl alcohol dehydrogenase (CAD) Gene family Function Monolignols Populus tomentosa Sinapyl alcohol dehydrogenase (SAD) 

Notes

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, and Grants J1103516, J1310005 and ITR 13047 for the Basic Science Basement Facility Buildup and Talent Training Program Project from National Natural Science Foundation of China (NSFC) to BJFU.

Supplementary material

425_2014_2128_MOESM1_ESM.xlsx (11 kb)
Online Resource Table S1 Primers used for PCR (XLSX 11 kb)
425_2014_2128_MOESM2_ESM.xlsx (11 kb)
Online Resource Table S2 Homology matrix of 13 sequences (DNAman7.0) (XLSX 10 kb)
425_2014_2128_MOESM3_ESM.xlsx (16 kb)
Online Resource Table S3 Blast results for PtoCAD/CAD-like genes with probes (XLSX 15 kb)
425_2014_2128_MOESM4_ESM.xlsx (17 kb)
Online Resource Table S4 List of plant genes used in CAD gene phylogenetic analyses (XLSX 17 kb)
425_2014_2128_MOESM5_ESM.pdf (399 kb)
Supplementary material 5 (PDF 398 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Nan Chao
    • 1
  • Shu-Xin Liu
    • 1
  • Bing-Mei Liu
    • 1
  • Ning Li
    • 1
  • Xiang-Ning Jiang
    • 1
    • 2
    • 3
  • Ying Gai
    • 1
    • 2
    Email author
  1. 1.College of Biological Sciences and BiotechnologyBeijing Forestry UniversityBeijingPeople’s Republic of China
  2. 2.The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of Chinese Forestry AdministrationBeijingPeople’s Republic of China
  3. 3.National Engineering Laboratory for Tree BreedingBeijingPeople’s Republic of China

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