@article{peng_tong_lee_wang_yu_huang_wen_makarem_pang_hinjan_et al._2023, title={Overexpression of a gibberellin 20-oxidase gene in poplar xylem led to an increase in the size of nanocellulose fibrils and improved paper properties}, volume={314}, ISSN={["1879-1344"]}, DOI={10.1016/j.carbpol.2023.120959}, abstractNote={Cellulose, the major component of secondary cell walls, is the most abundant renewable long-chain polymer on earth. Nanocellulose has become a prominent nano-reinforcement agent for polymer matrices in various industries. We report the generation of transgenic hybrid poplar overexpressing the Arabidopsis gibberellin 20-oxidase1 gene driven by a xylem-specific promoter to increase gibberellin (GA) biosynthesis in wood. X-ray diffraction (XRD) and sum frequency generation spectroscopic (SFG) analyses showed that cellulose in transgenic trees was less crystalline, but the crystal size was larger. The nanocellulose fibrils prepared from transgenic wood had an increased size compared to those from wild type. When such fibrils were used as a reinforcing agent in sheet paper preparation, the mechanical strength of the paper was significantly enhanced. Engineering the GA pathway can therefore affect nanocellulose properties, providing a new strategy for expanding nanocellulose applications.}, journal={CARBOHYDRATE POLYMERS}, author={Peng, Xiaopeng and Tong, Botong and Lee, Jongcheol and Wang, Kun and Yu, Xiaojuan and Huang, Xiong and Wen, Jialong and Makarem, Mohamadamin and Pang, Hongying and Hinjan, Subin and et al.}, year={2023}, month={Aug} }
 @article{wang_dai_pang_cheng_huang_li_yan_lu_wei_sederoff_et al._2021, title={BEL1-like Homeodomain Protein BLH6a Is a Negative Regulator of CAl5H2 in Sinapyl Alcohol Monolignol Biosynthesis in Poplar}, volume={12}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2021.695223}, abstractNote={Lignin is one of the major components of xylem cell walls in tree stems. The lignin in the wood of most flowering plants (dicotyledonous angiosperms) is typically polymerized from three monolignol precursors, coniferyl alcohol, sinapyl alcohol, and p-coumaroyl alcohol, resulting in guaiacyl (G), syringyl (S), and hydroxyphenyl (H) subunits, respectively. In this study, we focus on the transcriptional regulation of a coniferaldehyde 5-hydroxylase (CAld5H2) gene, which encodes a key enzyme for sinapyl alcohol biosynthesis. We carried out a yeast one-hybrid (Y1H) screen to identify candidate upstream transcription factors (TFs) regulating CAld5H2. We obtained 12 upstream TFs as potential regulators of CAld5H2. One of these TF genes, BLH6a, encodes a BEL1-like homeodomain (BLH) protein and negatively regulated the CAld5H2 promoter activity. The direct regulation of CAld5H2 promoter by BLH6a was supported by chromatin immunoprecipitation–quantitative polymerase chain reaction (ChIP–qPCR) and dominant repression of BLH6a in transgenic plants. Luciferase complementation imaging analyses showed extensive protein–protein interactions among these 12 TFs. We propose that BLH6a is a negative regulator of CAld5H2, which acts through combinatorial regulation of multiple TFs for sinapyl alcohol (S monolignol) biosynthesis in poplar.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Wang, Qiao and Dai, Xinren and Pang, Hongying and Cheng, Yanxia and Huang, Xiong and Li, Hui and Yan, Xiaojing and Lu, Fachuang and Wei, Hairong and Sederoff, Ronald R. and et al.}, year={2021}, month={Jun} }
 @article{wang_dai_pang_cheng_huang_li_yan_lu_wei_sederoff_et al._2021, title={BEL1-like Homeodomain Protein BLH6a Is a Negative Regulator of CAld5H2 in Sinapyl Alcohol Monolignol Biosynthesis in Poplar (vol 12,& nbsp;695223, 2021)}, volume={12}, ISSN={["1664-462X"]}, DOI={10.3389/fpls.2021.761291}, abstractNote={[This corrects the article DOI: 10.3389/fpls.2021.695223.].}, journal={FRONTIERS IN PLANT SCIENCE}, author={Wang, Qiao and Dai, Xinren and Pang, Hongying and Cheng, Yanxia and Huang, Xiong and Li, Hui and Yan, Xiaojing and Lu, Fachuang and Wei, Hairong and Sederoff, Ronald R. and et al.}, year={2021}, month={Sep} }
 @article{yan_liu_kim_liu_huang_yang_lin_chen_yang_wang_et al._2019, title={CAD1 and CCR2 protein complex formation in monolignol biosynthesis in Populus trichocarpa}, volume={222}, ISSN={["1469-8137"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85055556739&partnerID=MN8TOARS}, DOI={10.1111/nph.15505}, abstractNote={Summary}, number={1}, journal={NEW PHYTOLOGIST}, author={Yan, Xiaojing and Liu, Jie and Kim, Hoon and Liu, Baoguang and Huang, Xiong and Yang, Zhichang and Lin, Ying-Chung Jimmy and Chen, Hao and Yang, Chenmin and Wang, Jack P. and et al.}, year={2019}, month={Apr}, pages={244–260} }
 @article{li_yeh_yang_song_chen_sederoff_chiang_2015, title={Populus trichocarpa}, volume={1224}, ISBN={["978-1-4939-1657-3"]}, ISSN={["1064-3745"]}, DOI={10.1007/978-1-4939-1658-0_28}, abstractNote={Populus trichocarpa Nisqually-1 is a clone of black cottonwood that is widely used as a model woody plant. It was the first woody plant to have a full genome sequence and remains today as the model for growth, metabolism, development, and adaptation for all woody dicotyledonous plants. It is one of the best-annotated plant genomes available. It is also currently studied to improve bioenergy feedstocks and to learn about responses to environmental variation that may result from climate change. It is the best characterized woody plant for lignin biosynthesis. In spite of its role as a model woody plant, many important genetic applications have been limited because it was particularly difficult for DNA transformation. The ability to transform P. trichocarpa is a central component of a systems biology approach to the study of metabolic and developmental processes, where in combination with genome and transcriptome sequencing, all the expressed genes for specific pathways can be defined, cloned, and characterized for biological function. We previously reported on a method for Agrobacterium-mediated genetic transformation in P. trichocarpa(Song et al. Plant Cell Physiol 47: 1582-1589, 2006). Since then, we have optimized the protocol based on many experiments that varied in tissue manipulation, media, DNA constructs and Agrobacterium strains. A modified step-by-step protocol for Agrobacterium-mediated transformation of stem explants is described here. The health of the tissue explants and the time of cocultivation are among the critical steps in the protocol for successful transformation. This updated protocol should be helpful to many laboratories that are currently carrying out P. trichocarpa transformation. It should also encourage many labs that have not yet had success with P. trichocarpa to try again.}, journal={AGROBACTERIUM PROTOCOLS, VOLUME 2, THIRD EDITION}, author={Li, Quanzi and Yeh, Ting-Feng and Yang, Chenmin and Song, Jingyuan and Chen, Zenn-Zong and Sederoff, Ronald R. and Chiang, Vincent L.}, year={2015}, pages={357–363} }
 @article{li_lin_li_shi_lin_chen_chuang_qu_sederoff_chiang_2014, title={A robust chromatin immunoprecipitation protocol for studying transcription factor-DNA interactions and histone modifications in wood-forming tissue}, volume={9}, ISSN={["1750-2799"]}, DOI={10.1038/nprot.2014.146}, abstractNote={Woody cells and tissues are recalcitrant to standard chromatin immunoprecipitation (ChIP) procedures. However, we recently successfully implemented ChIP in wood-forming tissue of the model woody plant Populus trichocarpa. Here we provide the detailed ChIP protocol optimized for wood-forming tissue that we used in those studies. By using stem-differentiating xylem (SDX; a wood-forming tissue), we identified all steps that were ineffective in standard ChIP protocols and systematically modified them to develop and optimize a robust ChIP protocol. The protocol includes tissue collection, cross-linking, nuclear isolation, chromatin extraction, DNA fragmentation, immunoprecipitation, DNA purification and sequence analysis. The protocol takes 2.5 d to complete and allows a robust 8-10-fold enrichment of transcription factor (TF)-bound genomic fragments (~150 ng/g of SDX) over nonspecific DNAs. The enriched DNAs are of high quality and can be used for subsequent PCR and DNA-seq analyses. We used this protocol to identify genome-wide specific TF-DNA interactions during wood formation and histone modifications associated with regulation of wood formation. Our protocol, which may be suitable for many tissue types, is so far the only working ChIP system for wood-forming tissue.}, number={9}, journal={NATURE PROTOCOLS}, author={Li, Wei and Lin, Ying-Chung and Li, Quanzi and Shi, Rui and Lin, Chien-Yuan and Chen, Hao and Chuang, Ling and Qu, Guan-Zheng and Sederoff, Ronald R. and Chiang, Vincent L.}, year={2014}, month={Sep}, pages={2180–2193} }
 @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{min_li_chiang_jameel_chang_lucia_2014, title={nnThe influence of lignin-carbohydrate complexes on the cellulase-mediated saccharification I: Transgenic black cottonwood (western balsam poplar, California poplar) P-trichocarpa including the xylan down-regulated and the lignin down-regulated lines}, volume={119}, ISSN={["1873-7153"]}, DOI={10.1016/j.fuel.2013.11.047}, abstractNote={The influence of the putative lignin–carbohydrate complexes (LCCs) on enzymatic saccharification was elucidated for the first time by examining two groups of transgenic black cottonwood (P. trichocarpa) comprised of the lignin down-regulated and the xylan down-regulated lines. Any adventitious contaminants that could affect the characterization of LCCs and the enzymatic saccharification were removed by performing a thorough extraction on the samples. The crude milled wood lignin was subsequently isolated from which the phenyl glycoside, benzyl ether and γ-ester linkages representative of the LCCs were identified and quantified with the combination of 13C and 1H–13C Heteronuclear Single Quantum Coherence (HSQC) NMR. The result indicated that the samples showed different levels of the three LCC linkages, depending on the xylan and/or lignin content. The correlation between the LCCs and enzymatic saccharification nearly conclusively demonstrated that the LCCs accounting for the recalcitrance of lignocellulosic biodegradation.}, journal={FUEL}, author={Min, Dou-yong and Li, Quanzi and Chiang, Vincent and Jameel, Hasan and Chang, Hou-min and Lucia, Lucian}, year={2014}, month={Mar}, pages={207–213} }
 @article{liu_peng_li_sun_chiang_sederoff_2013, title={High-level gene expression in differentiating xylem of tobacco driven by a 2.0 kb Poplar COMT2 promoter and a 4 x 35S enhancer}, volume={30}, ISSN={["1342-4580"]}, DOI={10.5511/plantbiotechnology.13.0213a}, abstractNote={Promoter constructs with high levels of xylem specific expression are needed to obtain efficient expression of candidate genes, microRNAs (miRNAs) and artificial microRNAs (amiRNAs) for the genetic modification of wood properties. The gene for caffeic acid O-methytransferase (PtrCOMT2) has the second most abundant transcript level of all the genes in monolignol biosynthesis in Populus trichocarpa and a high level of specificity in differentiating xylem. To characterize the PtrCOMT2 promoter, we cloned a short (2.0 kb) and a long (3.3 kb) promoter segment and compared their expression using GUS as a reporter gene in the differentiating xylem of Nicotiana tabacum. Both the 2.0 kb and the 3.3 kb promoter segments showed high specificity for differentiating xylem in this heterologous system. GUS activity increased as much as 5 times when the 4×35S enhancer was inserted in front of the 2.0 kb promoter, but GUS activity was only increased 2 times when the enhancer was inserted behind the promoter. The enhancer inserted upstream reduced the expression of the 3.3 kb promoter. While expression of some of the enhancer-plus-promoter constructs increased expression, there was a loss of specificity.}, number={2}, journal={PLANT BIOTECHNOLOGY}, author={Liu, Enying and Peng, Shaobing and Li, Quanzi and Sun, Ying-Hsuan and Chiang, Vincent L. and Sederoff, Ronald R.}, year={2013}, pages={191–198} }
 @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{lu_li_wei_chang_tunlaya-anukit_kim_liu_song_sun_yuan_et al._2013, title={Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa}, volume={110}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/PNAS.1308936110}, DOI={10.1073/pnas.1308936110}, abstractNote={
            Laccases, as early as 1959, were proposed to catalyze the oxidative polymerization of monolignols. Genetic evidence in support of this hypothesis has been elusive due to functional redundancy of laccase genes. An
            Arabidopsis
            double mutant demonstrated the involvement of laccases in lignin biosynthesis. We previously identified a subset of laccase genes to be targets of a microRNA (miRNA) ptr-miR397a in
            Populus trichocarpa
            . To elucidate the roles of ptr-miR397a and its targets, we characterized the laccase gene family and identified 49 laccase gene models, of which 29 were predicted to be targets of ptr-miR397a. We overexpressed
            Ptr-MIR397a
            in transgenic
            P. trichocarpa
            . In each of all nine transgenic lines tested, 17
            PtrLAC
            s were down-regulated as analyzed by RNA-seq. Transgenic lines with severe reduction in the expression of these laccase genes resulted in an ∼40% decrease in the total laccase activity. Overexpression of
            Ptr-MIR397a
            in these transgenic lines also reduced lignin content, whereas levels of all monolignol biosynthetic gene transcripts remained unchanged. A hierarchical genetic regulatory network (GRN) built by a bottom-up graphic Gaussian model algorithm provides additional support for a role of ptr-miR397a as a negative regulator of laccases for lignin biosynthesis. Full transcriptome–based differential gene expression in the overexpressed transgenics and protein domain analyses implicate previously unidentified transcription factors and their targets in an extended hierarchical GRN including ptr-miR397a and laccases that coregulate lignin biosynthesis in wood formation. Ptr-miR397a, laccases, and other regulatory components of this network may provide additional strategies for genetic manipulation of lignin content.
          }, number={26}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Lu, S. and Li, Q. and Wei, H. and Chang, M.-J. and Tunlaya-Anukit, S. and Kim, H. and Liu, J. and Song, J. and Sun, Y.-H. and Yuan, L. and et al.}, year={2013}, month={Jun}, pages={10848–10853} }
 @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{lin_li_sun_kumari_wei_li_tunlaya-anukit_sederoff_chiang_2013, title={SND1 Transcription Factor-Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa}, volume={25}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.113.117697}, abstractNote={Novel methods were developed and demonstrated for the discovery of genetic regulatory networks in wood-forming tissues. Transfection of protoplasts from differentiating xylem with the transcription factor gene Ptr-SND1-B1 and novel computational analysis revealed a three-level hierarchical genetic regulatory network that was verified by ChIP and Ptr-SND1-B1 overexpression in transgenic plants. Wood is an essential renewable raw material for industrial products and energy. However, knowledge of the genetic regulation of wood formation is limited. We developed a genome-wide high-throughput system for the discovery and validation of specific transcription factor (TF)–directed hierarchical gene regulatory networks (hGRNs) in wood formation. This system depends on a new robust procedure for isolation and transfection of Populus trichocarpa stem differentiating xylem protoplasts. We overexpressed Secondary Wall-Associated NAC Domain 1s (Ptr-SND1-B1), a TF gene affecting wood formation, in these protoplasts and identified differentially expressed genes by RNA sequencing. Direct Ptr-SND1-B1–DNA interactions were then inferred by integration of time-course RNA sequencing data and top-down Graphical Gaussian Modeling–based algorithms. These Ptr-SND1-B1-DNA interactions were verified to function in differentiating xylem by anti-PtrSND1-B1 antibody-based chromatin immunoprecipitation (97% accuracy) and in stable transgenic P. trichocarpa (90% accuracy). In this way, we established a Ptr-SND1-B1–directed quantitative hGRN involving 76 direct targets, including eight TF and 61 enzyme-coding genes previously unidentified as targets. The network can be extended to the third layer from the second-layer TFs by computation or by overexpression of a second-layer TF to identify a new group of direct targets (third layer). This approach would allow the sequential establishment, one two-layered hGRN at a time, of all layers involved in a more comprehensive hGRN. Our approach may be particularly useful to study hGRNs in complex processes in plant species resistant to stable genetic transformation and where mutants are unavailable.}, number={11}, journal={PLANT CELL}, author={Lin, Ying-Chung and Li, Wei and Sun, Ying-Hsuan and Kumari, Sapna and Wei, Hairong and Li, Quanzi and Tunlaya-Anukit, Sermsawat and Sederoff, Ronald R. and Chiang, Vincent L.}, year={2013}, month={Nov}, pages={4324–4341} }
 @article{loziuk_wang_li_sederoff_chiang_muddiman_2013, title={Understanding the Role of Proteolytic Digestion on Discovery and Targeted Proteomic Measurements Using Liquid Chromatography Tandem Mass Spectrometry and Design of Experiments}, volume={12}, ISSN={["1535-3907"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84890109136&partnerID=MN8TOARS}, DOI={10.1021/pr4008442}, abstractNote={Workflows in bottom-up proteomics have traditionally implemented the use of proteolysis during sample preparation; enzymatic digestion is most commonly performed using trypsin. This results in the hydrolysis of peptide bonds forming tryptic peptides, which can then be subjected to LC-MS/MS analysis. While the structure, specificity, and kinetics of trypsin are well characterized, a lack of consensus and understanding has remained regarding fundamental parameters critical to obtaining optimal data from a proteomics experiment. These include the type of trypsin used, pH during digestion, incubation temperature as well as enzyme-to-substrate ratio. Through the use of design of experiments (DOE), we optimized these parameters, resulting in deeper proteome coverage and a greater dynamic range of measurement. The knowledge gained from optimization of a discovery-based proteomics experiment was applied to targeted LC-MS/MS experiments using protein cleavage-isotope dilution mass spectrometry for absolute quantification. We demonstrated the importance of these digest parameters with respect to our limit of detection as well as our ability to acquire more accurate quantitative measurements. Additionally, we were able to quantitatively account for peptide decay observed in previous studies, caused by nonspecific activity of trypsin. The tryptic digest optimization described here has eliminated this previously observed peptide decay as well as provided a greater understanding and standardization for a common but critical sample treatment used across the field of proteomics.}, number={12}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Loziuk, Philip L. and Wang, Jack and Li, Quanzi and Sederoff, Ronald R. and Chiang, Vincent L. and Muddiman, David C.}, year={2013}, month={Dec}, pages={5820–5829} }
 @article{liu_shi_li_sederoff_chiang_2012, title={A standard reaction condition and a single HPLC separation system are sufficient for estimation of monolignol biosynthetic pathway enzyme activities}, volume={236}, ISSN={["0032-0935"]}, DOI={10.1007/s00425-012-1688-9}, abstractNote={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.}, number={3}, journal={PLANTA}, author={Liu, Jie and Shi, Rui and Li, Quanzi and Sederoff, Ronald R. and Chiang, Vincent L.}, year={2012}, month={Sep}, pages={879–885} }
 @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{li_lin_sun_song_chen_zhang_sederoff_chiang_2012, title={Splice variant of the SND1 transcription factor is a dominant negative of SND1 members and their regulation in Populus trichocarpa}, volume={109}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1212977109}, abstractNote={
            Secondary Wall-Associated NAC Domain 1s (SND1s) are transcription factors (TFs) known to activate a cascade of
            TF
            and pathway genes affecting secondary cell wall biosynthesis (xylogenesis) in
            Arabidopsis
            and poplars. Elevated SND1 transcriptional activation leads to ectopic xylogenesis and stunted growth. Nothing is known about the upstream regulators of
            SND1
            . Here we report the discovery of a stem-differentiating xylem (SDX)-specific alternative
            SND1
            splice variant,
            PtrSND1
            -
            A2
            
              IR
            
            , that acts as a dominant negative of SND1 transcriptional network genes in
            Populus trichocarpa
            .
            PtrSND1
            -
            A2
            
              IR
            
            derives from
            PtrSND1-A2
            , one of the four fully spliced
            PtrSND1
            gene family members (
            PtrSND1
            -
            A1
            , -
            A2
            , -
            B1
            , and -
            B2
            ). Each full-size PtrSND1 activates its own gene, and all four full-size members activate a common
            MYB
            gene (
            PtrMYB021
            ). PtrSND1-A2
            IR
            represses the expression of its
            PtrSND1
            member genes and
            PtrMYB021
            . Repression of the autoregulation of a TF family by its only splice variant has not been previously reported in plants. PtrSND1-A2
            IR
            lacks DNA binding and transactivation abilities but retains dimerization capability. PtrSND1-A2
            IR
            is localized exclusively in cytoplasmic foci. In the presence of any full-size PtrSND1 member, PtrSND1-A2
            IR
            is translocated into the nucleus exclusively as a heterodimeric partner with full-size PtrSND1s. Our findings are consistent with a model in which the translocated PtrSND1-A2
            IR
            lacking DNA-binding and transactivating abilities can disrupt the function of full-size PtrSND1s, making them nonproductive through heterodimerization, and thereby modulating the SND1 transcriptional network. PtrSND1-A2
            IR
            may contribute to transcriptional homeostasis to avoid deleterious effects on xylogenesis and plant growth.
          }, number={36}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Li, Quanzi and Lin, Ying-Chung and Sun, Ying-Hsuan and Song, Jian and Chen, Hao and Zhang, Xing-Hai and Sederoff, Ronald R. and Chiang, Vincent L.}, year={2012}, month={Sep}, pages={14699–14704} }
 @article{min_li_jameel_chiang_chang_2012, title={The Cellulase-Mediated Saccharification on Wood Derived from Transgenic Low-Lignin Lines of Black Cottonwood (Populus trichocarpa)}, volume={168}, ISSN={["1559-0291"]}, DOI={10.1007/s12010-012-9833-2}, abstractNote={Downregulated lignin transgenic black cottonwood (Populus trichocarpa) was used to elucidate the effect of lignin and xylan content on enzymatic saccharification. The lignin contents of three transgenic samples (4CL1-1, 4CL1-4, and CH8-1-4) were 19.3, 16.7, and 15.0 %, respectively, as compared with the wild type (21.3 %). The four pretreatments were dilute acid (0.1 % sulfuric acid, 185 °C, 30 min), green liquor (6 % total titratable alkali, 25 % sulfidity based on TTA, 185 °C, and 15 min.), autohydrolysis (185 °C, 30 min), and ozone delignification (25 °C, 30 min). Following the pretreatment, enzymatic saccharification was carried out using an enzyme charge of 5 FPU/g of substrates. The removal of lignin and hemicellulose varies with both the types of pretreatments and the lignin content of the transgenic trees. Due to the greatest removal of lignin, green liquor induced the highest sugar production and saccharification efficiency, followed by acid, ozone, and autohydrolysis in descending order. The results indicated that lignin is the main recalcitrance of biomass degradation. At a given lignin content, pretreatment with ozone delignification had lower saccharification efficiency than the other pretreatment methods due to higher xylan content.}, number={4}, journal={APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY}, author={Min, Douyong and Li, Quanzi and Jameel, Hasan and Chiang, Vincent and Chang, Hou-min}, year={2012}, month={Oct}, pages={947–955} }
 @article{li_min_wang_peszlen_horvath_horvath_nishimura_jameel_chang_chiang_2011, title={Down-regulation of glycosyltransferase 8D genes in Populus trichocarpa caused reduced mechanical strength and xylan content in wood}, volume={31}, ISSN={["1758-4469"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-79953831391&partnerID=MN8TOARS}, DOI={10.1093/treephys/tpr008}, abstractNote={Members of glycosyltransferase protein families GT8, GT43 and GT47 are implicated in the biosynthesis of xylan in the secondary cell walls of Arabidopsis. The Arabidopsis mutant irx8 has a 60% reduction in xylan. However, over-expression of an ortholog of Arabidopsis IRX8, poplar PoGT8D, in Arabidopsis irx8 mutant could not restore xylan synthesis. The functions of tree GT8D genes remain unclear. We identified two GT8 gene homologs, PtrGT8D1 and PtrGT8D2, in Populus trichocarpa. They are the only two GT8D members and are abundantly and specifically expressed in the differentiating xylem of P. trichocarpa. PtrGT8D1 transcript abundance was >7 times that of PtrGT8D2. To elucidate the genetic function of GT8D in P. trichocarpa, the expression of PtrGT8D1 and PtrGT8D2 was simultaneously knocked down through RNAi. Four transgenic lines had 85-94% reduction in transcripts of PtrGT8D1 and PtrGT8D2, resulting in 29-36% reduction in stem wood xylan content. Xylan reduction had essentially no effect on cellulose quantity but caused an 11-25% increase in lignin. These transgenics exhibit a brittle wood phenotype, accompanied by increased vessel diameter and thinner fiber cell walls in stem xylem. Stem modulus of elasticity and modulus of rupture were reduced by 17-29% and 16-23%, respectively, and were positively correlated with xylan content but negatively correlated with lignin quantity. These results suggest that PtrGT8Ds play key roles in xylan biosynthesis in wood. Xylan may be a more important factor than lignin affecting the stiffness and fracture strength of wood.}, number={2}, journal={TREE PHYSIOLOGY}, publisher={Oxford University Press (OUP)}, author={Li, Quanzi and Min, Douyong and Wang, Jack Peng-Yu and Peszlen, Ilona and Horvath, Laszlo and Horvath, Balazs and Nishimura, Yufuko and Jameel, Hasan and Chang, Hou-Min and Chiang, Vincent L.}, year={2011}, month={Feb}, pages={226–236} }
 @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} }