@article{lin_sun_song_chen_shi_yang_liu_tunlaya-anukit_liu_loziuk_et al._2021, title={Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa}, volume={12}, ISSN={["1664-462X"]}, url={http://europepmc.org/abstract/med/34691108}, DOI={10.3389/fpls.2021.727932}, abstractNote={Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. InPopulus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation ofPtrHCTsreduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenicP. trichocarpa. The Ptr4CL/PtrHCT interactions were then validatedin vivousing biomolecular fluorescence complementation (BiFC) and protein pull-down assays inP. trichocarpaSDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation inP. trichocarpa.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Lin, Chien-Yuan and Sun, Yi and Song, Jina and Chen, Hsi-Chuan and Shi, Rui and Yang, Chenmin and Liu, Jie and Tunlaya-Anukit, Sermsawat and Liu, Baoguang and Loziuk, Philip L. and et al.}, year={2021}, month={Oct} }
 @article{lin_wang_li_chen_liu_loziuk_song_williams_muddiman_sederoff_et al._2015, title={4-Coumaroyl and Caffeoyl Shikimic Acids Inhibit 4-Coumaric Acid: Coenzyme A Ligases and Modulate Metabolic Flux for 3-Hydroxylation in Monolignol Biosynthesis of Populus trichocarpa}, volume={8}, ISSN={["1752-9867"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84925201417&partnerID=MN8TOARS}, DOI={10.1016/j.molp.2014.12.003}, abstractNote={Downregulation of 4-coumaric acid:coenzyme A ligase (4CL) can reduce lignin content in a number of plant species. In lignin precursor (monolignol) biosynthesis during stem wood formation in Populus trichocarpa, two enzymes, Ptr4CL3 and Ptr4CL5, catalyze the coenzyme A (CoA) ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. CoA ligation of 4-coumaric acid is essential for the 3-hydroxylation of 4-coumaroyl shikimic acid. This hydroxylation results from sequential reactions of 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) and 4-coumaric acid 3-hydroxylase 3 (PtrC3H3). Alternatively, 3-hydroxylation of 4-coumaric acid to caffeic acid may occur through an enzyme complex of cinnamic acid 4-hydroxylase 1 and 2 (PtrC4H1 and PtrC4H2) and PtrC3H3. We found that 4-coumaroyl and caffeoyl shikimic acids are inhibitors of Ptr4CL3 and Ptr4CL5. 4-Coumaroyl shikimic acid strongly inhibits the formation of 4-coumaroyl-CoA and caffeoyl-CoA. Caffeoyl shikimic acid inhibits only the formation of 4-coumaroyl-CoA. 4-Coumaroyl and caffeoyl shikimic acids both act as competitive and uncompetitive inhibitors. Metabolic flux in wild-type and PtrC3H3 downregulated P. trichocarpa transgenics has been estimated by absolute protein and metabolite quantification based on liquid chromatography–tandem mass spectrometry, mass action kinetics, and inhibition equations. Inhibition by 4-coumaroyl and caffeoyl shikimic acids may play significant regulatory roles when these inhibitors accumulate.}, number={1}, journal={MOLECULAR PLANT}, author={Lin, Chien-Yuan and Wang, Jack P. and Li, Quanzi and Chen, Hsi-Chuan and Liu, Jie and Loziuk, Philip and Song, Jina and Williams, Cranos and Muddiman, David C. and Sederoff, Ronald R. and et al.}, year={2015}, month={Jan}, pages={176–187} }
 @article{lin_li_chen_li_sun_shi_lin_wang_chen_chuang_et al._2014, title={A simple improved-throughput xylem protoplast system for studying wood formation}, volume={9}, ISSN={["1750-2799"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84907026654&partnerID=MN8TOARS}, DOI={10.1038/nprot.2014.147}, abstractNote={Isolated protoplasts serve as a transient expression system that is highly representative of stable transgenics in terms of transcriptome responses. They can also be used as a cellular system to study gene transactivation and nucleocytoplasmic protein trafficking. They are particularly useful for systems studies in which stable transgenics and mutants are unavailable. We present a protocol for the isolation and transfection of protoplasts from wood-forming tissue, the stem-differentiating xylem (SDX), in the model woody plant Populus trichocarpa. The method involves tissue preparation, digestion of SDX cell walls, protoplast isolation and DNA transfection. Our approach is markedly faster and provides better yields than previous protocols; small (milligrams)- to large (20 g)-scale SDX preparations can be achieved in ~60 s, with isolation of protoplasts and their subsequent transfection taking ~50 min. Up to ten different samples can be processed simultaneously in this time scale. Our protocol gives a high yield (~2.5 × 10(7) protoplasts per g of SDX) of protoplasts sharing 96% transcriptome identity with intact SDX.}, number={9}, journal={NATURE PROTOCOLS}, author={Lin, Ying-Chung and Li, Wei and Chen, Hao and Li, Quanzi and Sun, Ying-Hsuan and Shi, Rui and Lin, Chien-Yuan and Wang, Jack P. and Chen, Hsi-Chuan and Chuang, Ling and et al.}, year={2014}, month={Sep}, pages={2194–2205} }
 @article{chen_song_wang_lin_ducoste_shuford_liu_li_shi_nepomuceno_et al._2014, title={Systems Biology of Lignin Biosynthesis in Populus trichocarpa: Heteromeric 4-Coumaric Acid:Coenzyme A Ligase Protein Complex Formation, Regulation, and Numerical Modeling}, volume={26}, ISSN={1040-4651 1532-298X}, url={http://dx.doi.org/10.1105/tpc.113.119685}, DOI={10.1105/tpc.113.119685}, abstractNote={This work shows that 4CL, an enzyme in monolignol biosynthesis, is found as a heterotetrameric complex of two isoforms in Populus trichocarpa. The activity of the heterotetramer can be described by a mathematical model that explains the effects of each isoform with mixtures of substrates and three types of inhibition, providing insights into the regulation of metabolic flux for this pathway. As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein–protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.}, number={3}, journal={The Plant Cell}, publisher={Oxford University Press (OUP)}, author={Chen, Hsi-Chuan and Song, Jina and Wang, Jack P. and Lin, Ying-Chung and Ducoste, Joel and Shuford, Christopher M. and Liu, Jie and Li, Quanzi and Shi, Rui and Nepomuceno, Angelito and et al.}, year={2014}, month={Mar}, pages={876–893} }
 @article{chen_song_williams_shuford_liu_wang_li_shi_gokce_ducoste_et al._2013, title={Monolignol Pathway 4-Coumaric Acid: Coenzyme A Ligases in Populus trichocarpa: Novel Specificity, Metabolic Regulation, and Simulation of Coenzyme A Ligation Fluxes}, volume={161}, ISSN={["0032-0889"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84874626790&partnerID=MN8TOARS}, DOI={10.1104/pp.112.210971}, abstractNote={Abstract}, number={3}, journal={PLANT PHYSIOLOGY}, author={Chen, Hsi-Chuan and Song, Jina and Williams, Cranos M. and Shuford, Christopher M. and Liu, Jie and Wang, Jack P. and Li, Quanzi and Shi, Rui and Gokce, Emine and Ducoste, Joel and et al.}, year={2013}, month={Mar}, pages={1501–1516} }
 @article{shi_shuford_wang_sun_yang_chen_tunlaya-anukit_li_liu_muddiman_et al._2013, title={Regulation of phenylalanine ammonia-lyase (PAL) gene family in wood forming tissue of Populus trichocarpa}, volume={238}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84882877816&partnerID=MN8TOARS}, DOI={10.1007/s00425-013-1905-1}, abstractNote={Phenylalanine ammonia-lyase (PAL) catalyzes the initial step of phenylpropanoid biosynthesis in plants. Five PAL genes (PtrPAL1 to 5) have been identified in Populus trichocarpa. These genes are classified into two subgroups according to their transcript sequence similarity and tissue specificity. However, the regulation of these genes and their protein functions are not well understood. In this study, enzymatic properties of each PtrPALs were characterized based on their recombinant proteins expressed in E.coli. Subcellular localizations of each PtrPALs in stem wood forming tissue were investigated and individual PtrPAL protein abundances in cytosol and membrane protein fractions were measured using protein cleavage-isotope dilution mass spectrometry (PC-IDMS). Protein/mRNA ratios of PtrPALs were further verified using RNA-Seq and gel-enhanced liquid chromatography mass spectrometry (GeLC-MS). All PtrPALs have similar catalytic properties for the deamination of L-phenylalanine, their major substrate. All PtrPALs have similar subcellular locations in stem wood forming tissue, with major amount in the cytosol (93-96 %) and less in the membrane (4-7 %). However, the protein/mRNA ratios of subgroup A (PtrPAL2, 4 and 5) are about five times that of subgroup B (PtrPAL1 and 3) in stem wood forming tissue, while all PtrPALs have similar transcript abundances. These results indicate a greater functional significance of subgroup A PtrPALs for stem wood formation, and highlight the role of gene post-transcriptional regulation.}, number={3}, journal={Planta}, author={Shi, R. and Shuford, C. M. and Wang, Jack P. and Sun, Y. H. and Yang, Z. C. and Chen, H. C. and Tunlaya-Anukit, S. and Li, Q. Z. and Liu, J. and Muddiman, David and et al.}, year={2013}, pages={487–497} }
 @article{shuford_li_sun_chen_wang_shi_sederoff_chiang_muddiman_2012, title={Comprehensive Quantification of Monolignol-Pathway Enzymes in Populus trichocarpa by Protein Cleavage Isotope Dilution Mass Spectrometry}, volume={11}, ISSN={1535-3893 1535-3907}, url={http://dx.doi.org/10.1021/pr300205a}, DOI={10.1021/pr300205a}, abstractNote={The economic value of wood/pulp from many tree species is largely dictated by the quantity and chemical properties of lignin, which is directly related to the composition and linkages of monolignols comprising the polymer. Although much is known regarding the monolignol biosynthetic pathway, our understanding is still deficient due to the lack of quantitative information at the proteomic level. We developed an assay based on protein cleavage isotope dilution mass spectrometry (PC-IDMS) for the determination of all potential, primary enzymes involved in the biosynthesis of monolignols and the peroxidases responsible for their polymerization to form lignin in the model tree species, Populus trichocarpa. Described is the identification of quantitative surrogate peptides through shotgun analysis of native and recombinant proteins, optimization of trypsin proteolysis using fractional factorial design of experiments, and development of a liquid chromatography-selected reaction monitoring method for specific detection of all targeted peptides. Of the 25 targeted enzymes, three were undetected in the normal xylem tissues, and all but two of the detectable species showed good day-to-day precision (CV < 10%). This represents the most comprehensive assay for quantification of proteins regulating monolignol biosynthesis and will lead to a better understanding of lignin formation at a systems level.}, number={6}, journal={Journal of Proteome Research}, publisher={American Chemical Society (ACS)}, author={Shuford, Christopher M. and Li, Quanzi and Sun, Ying-Hsuan and Chen, Hsi-Chuan and Wang, Jack and Shi, Rui and Sederoff, Ronald. R. and Chiang, Vincent L. and Muddiman, David C.}, year={2012}, month={May}, pages={3390–3404} }
 @article{wang_shuford_li_song_lin_sun_chen_williams_muddiman_sederoff_et al._2012, title={Functional redundancy of the two 5-hydroxylases in monolignol biosynthesis of Populus trichocarpa: LC-MS/MS based protein quantification and metabolic flux analysis}, volume={236}, ISSN={["1432-2048"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84865584315&partnerID=MN8TOARS}, DOI={10.1007/s00425-012-1663-5}, abstractNote={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.}, number={3}, journal={PLANTA}, publisher={Springer Science + Business Media}, author={Wang, Jack P. and Shuford, Christopher M. and Li, Quanzi and Song, Jina and Lin, Ying-Chung and Sun, Ying-Hsuan and Chen, Hsi-Chuan and Williams, Cranos M. and Muddiman, David C. and Sederoff, Ronald R. and et al.}, year={2012}, month={Sep}, pages={795–808} }
 @article{chen_li_shuford_liu_muddiman_sederoff_chiang_2011, title={Membrane protein complexes catalyze both 4-and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis}, volume={108}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1116416109}, abstractNote={
            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
            p
            -coumaric 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.
          }, number={52}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Chen, Hsi-Chuan and Li, Quanzi and Shuford, Christopher M. and Liu, Jie and Muddiman, David C. and Sederoff, Ronald R. and Chiang, Vincent L.}, year={2011}, month={Dec}, pages={21253–21258} }