Abstract
Lignin content and composition are largely determined by the composition and quantity of the monolignol precursors. Individual enzymes of the monolignol biosynthetic pathway determine the composition and quantity of monolignols. Monolignol biosynthesis in angiosperms is mediated by ten enzyme families. We developed a method using a total protein extract (soluble and microsomal) for the comprehensive and simultaneous analysis of these ten enzyme activities in a single target tissue, stem differentiating xylem (SDX) of Populus trichocarpa. As little as 300 mg fresh weight of SDX is sufficient for triplicate assays of all ten enzyme activities. To expand the effectiveness of the analysis, we quantified the reaction products directly by HPLC and developed a universal method that can separate the substrates and products of all enzymes. The specific activities measured with this simple approach are similar to those obtained with the optimum conditions previously established for each individual enzyme. This approach is applicable to the enzyme activity analysis for both P. trichocarpa (angiosperm) and Pinus taeda (gymnosperm) and is particularly useful when a large number of samples need to be analyzed for all monolignol biosynthetic enzymes.
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Abbreviations
- SDX:
-
Stem differentiating xylem
- PAL:
-
Phenylalanine ammonia-lyase
- C4H:
-
Cinnamate 4-hydroxylase
- C3H:
-
4-Coumarate 3-hydroxylase
- 4CL:
-
4-Coumarate: coenzyme A ligase
- HCT:
-
p-Hydroxycinnamoyl-CoA: quinate shikimate p-hydroxycinnamoyltransferase
- CCoAOMT:
-
Caffeoyl-CoA O-methyltransferase
- CAld5H:
-
Coniferaldehyde 5-hydroxylase
- COMT:
-
Caffeic acid O-methyltransferase
- CCR:
-
Cinnamoyl-CoA reductase
- CAD:
-
Cinnamyl alcohol dehydrogenase
- SAD:
-
Sinapyl alcohol dehydrogenase
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Acknowledgments
We thank Christopher M. Shuford and Dr. David C. Muddiman for mass spectrometry information of synthesized compounds; Hsi-Chuan Chen and Jack P. Wang for optimum condition information. This work was supported by National Science Foundation Plant Genome Research Program Grant DBI-0922391 (to V.L.C).
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A contribution to the Special Issue on Metabolic Plant Biology.
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Liu, J., Shi, R., Li, Q. et al. A standard reaction condition and a single HPLC separation system are sufficient for estimation of monolignol biosynthetic pathway enzyme activities. Planta 236, 879–885 (2012). https://doi.org/10.1007/s00425-012-1688-9
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DOI: https://doi.org/10.1007/s00425-012-1688-9