Plant Biotechnology
Online ISSN : 1347-6114
Print ISSN : 1342-4580
ISSN-L : 1342-4580
Original Papers
Characterization of three cinnamyl alcohol dehydrogenases from Carthamus tinctorius
Safendrri Komara RagamustariNaoko ShiraiwaTakefumi HattoriTomoyuki NakatsuboShiro SuzukiToshiaki Umezawa
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Supplementary material

2013 Volume 30 Issue 4 Pages 315-326

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Abstract

We report the isolation and characterization of three cDNAs encoding cinnamyl alcohol dehydrogenase (CAD) from Carthamus tinctorius (safflower). All three recombinant CADs were able to reduce coniferaldehyde and sinapaldehyde into coniferyl alcohol and sinapyl alcohol, respectively, and were designated as CtCAD1, CtCAD2, and CtCAD3. Phylogenetic analysis of CAD amino acid sequences and homology modeling revealed that CtCAD1 and CtCAD3 were closely related to the sinapaldehyde-specific aspen (Populus tremuloides) sinapyl alcohol dehydrogenase (PtreSAD). CtCAD2 was in a clade containing class I plant CADs. Gas chromatography-mass spectrometry-based kinetic analysis using two different substrates, coniferaldehyde and sinapaldehyde, indicated that the CtCADs showed no strong preference for either substrate. CtCAD2 has the highest catalytic efficiency (kcat/Km) (81.49 mM−1 min−1 and 95.3 mM−1 min−1 for coniferaldehyde and sinapaldehyde, respectively) compared with the other CtCADs. Inhibition kinetics showed that coniferaldehyde was a stronger inhibitor than sinapaldehyde for all CtCADs. Quantitative real-time PCR revealed that CtCAD2 was expressed at higher levels than CtCAD1 and CtCAD3 in all samples, except developing seeds at 3 days after flowering, where CtCAD1 had a higher expression level. In plant protein assays with coniferaldehyde and sinapaldehyde, plant protein extracted from seeds at 7 days after flowering, showed the highest specific activity. The product yields in plant protein assays were strongly correlated with gene expressions of CtCAD2 and CtCAD3 in the respective organs.

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© 2013 by Japanese Society for Plant Biotechnology
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