NADP-dependent cinnamyl alcohol dehydrogenases (CDH)

We have identified cinnamyl alcohol dehydrogenase enzymes in crude extracts from 2-8 cm primary roots. The spectrophotometric assay monitors the pseudo-zero order accumulation of NADPH in a reaction mixture composed as follows: enzyme extract, 0.1 mM NADP, 0.01 M cinnamyl alcohol from a 1.0 M stock in 100% MeOH, and 0.01 M NaPO4 at pH 7.3; the cinnamyl alcohol (0-C=C-C-OH) tends to come out of solution as an oil. Root extracts were subjected to electrophoresis in standard borate starch gels, cut and stained (D. Schwartz and T. Endo, Genetics 53:709, 1966). The stain was as the spectrophotometric reaction mixture except that cinnamyl alcohol was added last to 0.1 M and both p-nitrobluetetrazolium chloride and phenazine methosulfonate were present at 0.1 mM. We found several different banding patterns among the inbred lines which we tested (Std. S and Std. F from Schwartz). The Figure diagrams the electrophoretograms from three of these lines and some of the hybrids.

Figure.

By using the appropriate electrophoretic markers for Adh1 and Adh2, we found that ADH subunits are not involved in CDH activity.

We have performed the appropriate backcrosses and self-pollinations of the Std. S/Std. F and Std. F/B14 hybrids. An entire banding pattern segregates as a unit, although the F1's may not be the exact sum of their parental patterns. The majority of ears from these crosses segregated patterns consistent with Mendelian expectations: the Std. S pattern is allelic to the Std. F pattern, and the Std. F pattern is allelic to the B14 pattern. We call the responsible gene Cdh1.

The three CDH's present in the Std. S/Std. F hybrid were excluded from a Sephadex G150 column, suggesting that their MW are above 150,000. However, a Biogel P300 column separated all three CDH bands; the fastest electrophoretically was the largest at or near the 400,000 MW exclusion limit, with the slowest electrophoretically being considerably smaller; the intermediate isozyme in electrophoretic mobility is also intermediate in size. Such large sizes suggested to us that maize CDH is multimeric. The allelic differences which we have found among maize inbreds may reflect degrees of polymerization, etc. Such problems lie outside of this laboratory's current interest; we will supply data and lines on request.

Michael Freeling and James C. Woodman


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