Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis

Abstract

The hydroxylation of 4-and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V max /k m ) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/ C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts pcoumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis.

Monolignol Pathway 4-Coumaric Acid:Coenzyme A Ligases in Populus. trichocarpa: Novel Specificity, Metabolic Regulation, and Simulation of Coenzyme A Ligation Fluxes

“…1) to 4-coumaroyl CoA ester (8) and the other from caffeic acid (4) to caffeoyl CoA ester (11). These two acid-to-CoA ester fluxes are consistent with our recent discovery of two membrane-protein complex (C4H/C3H)-mediated 3-hydroxylation paths linking 4-coumaric acid (3) to caffeic acid (4) and 4-coumaroyl CoA ester (8) to caffeoyl CoA ester (11; Chen et al, 2011). While these two acid-to-CoA ester fluxes can be established by Ptr4CL3 alone (Table III; Fig.…”

“…None of them were proteins known in the monolignol biosynthetic pathway (Shi et al, 2010;Liu et al, 2012). Given that monolignol proteins are highly abundant in SDX (Shi et al, 2010;Chen et al, 2011;Liu et al, 2012;Shuford et al, 2012aShuford et al, , 2012b, if they were phosphorylated, they most likely would have been detected. A similar phosphopeptide-enrichment/mass spectrometry (MS) strategy for the analysis of the phosphoproteome of dormant terminal buds in Populus simonii 3 Populus nigra identified 151 phosphoproteins .…”

Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa

“…We focused on PtrMYB021 (POPTR_0009s05860), the ortholog of AtMYB46 (24). We overexpressed each of the five PtrSND1 genes in P. trichocarpa SDX protoplasts, using protocols that we recently developed (25). All four full-size PtrSND1s could induce a twofold to fourfold increase in abundance of endogenous PtrMYB021 transcripts in SDX protoplasts ( Fig.…”

SND1 Transcription Factor-Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa

“…The challenge is that woody plant tissues are generally resistant to protoplast isolation (Teulieres et al, 1991;Manders et al, 1992;Gomez-Maldonado et al, 2001;Sun et al, 2009;Tang et al, 2010;Chen et al, 2011;Li et al, 2012). Even for amenable woody plant tissues, protoplast isolation has never before been designed for yield and quality adequate for transgenesis, transcriptome and chromatin enrichment studies.…”