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Functional redundancy of the two 5-hydroxylases in monolignol biosynthesis of Populus trichocarpa: LC–MS/MS based protein quantification and metabolic flux analysis

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Abstract

Flowering plants have syringyl and guaiacyl subunits in lignin in contrast to the guaiacyl lignin in gymnosperms. The biosynthesis of syringyl subunits is initiated by coniferaldehyde 5-hydroxylase (CAld5H). In Populus trichocarpa there are two closely related CAld5H enzymes (PtrCAld5H1 and PtrCAld5H2) associated with lignin biosynthesis during wood formation. We used yeast recombinant PtrCAld5H1 and PtrCAld5H2 proteins to carry out Michaelis–Menten and inhibition kinetics with LC–MS/MS based absolute protein quantification. CAld5H, a monooxygenase, requires a cytochrome P450 reductase (CPR) as an electron donor. We cloned and expressed three P. trichocarpa CPRs in yeast and show that all are active with both CAld5Hs. The kinetic analysis shows both CAld5Hs have essentially the same biochemical functions. When both CAld5Hs are coexpressed in the same yeast membranes, the resulting enzyme activities are additive, suggesting functional redundancy and independence of these two enzymes. Simulated reaction flux based on Michaelis–Menten kinetics and inhibition kinetics confirmed the redundancy and independence. Subcellular localization of both CAld5Hs as sGFP fusion proteins expressed in P. trichocarpa differentiating xylem protoplasts indicate that they are endoplasmic reticulum resident proteins. These results imply that during wood formation, 5-hydroxylation in monolignol biosynthesis of P. trichocarpa requires the combined metabolic flux of these two CAld5Hs to maintain adequate biosynthesis of syringyl lignin. The combination of genetic analysis, absolute protein quantitation-based enzyme kinetics, homologous CPR specificity, SNP characterization, and ER localization provides a more rigorous basis for a comprehensive systems understanding of 5-hydroxylation in lignin biosynthesis.

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Abbreviations

SDX:

Stem differentiating xylem

CPR:

Cytochrome P450 reductase

CAld5H:

Coniferaldehyde 5-hydroxylase

PC-IDMS:

Protein cleavage isotope dilution mass spectrometry

ER:

Endoplasmic reticulum

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Acknowledgments

We thank Dr. Danièle Werck-Reichhart and Dr. Denis Pompon for the plasmid pYeDP60. This work was supported by National Science Foundation Plant Genome Research Program Grant DBI-0922391 (to V.L.C).

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Authors and Affiliations

  1. Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC, 27695, USA

    Jack P. Wang, Quanzi Li, Ying-Chung Lin, Ying-Hsuan Sun, Hsi-Chuan Chen, Ronald R. Sederoff & Vincent L. Chiang

  2. Department of Electrical and Computer Engineering, North Carolina State University, Raleigh, NC, 27695, USA

    Jina Song & Cranos M. Williams

  3. W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC, 27695, USA

    Christopher M. Shuford & David C. Muddiman

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  1. Jack P. Wang
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  11. Vincent L. Chiang
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Correspondence to Vincent L. Chiang.

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A contribution to the Special Issue on Metabolic Plant Biology.

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Wang, J.P., Shuford, C.M., Li, Q. et al. Functional redundancy of the two 5-hydroxylases in monolignol biosynthesis of Populus trichocarpa: LC–MS/MS based protein quantification and metabolic flux analysis. Planta 236, 795–808 (2012). https://doi.org/10.1007/s00425-012-1663-5

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