@article{yu_gao_li_li_chiang_li_zhou_2024, title={Ectopic Expression of <i>PtrLBD39</i> Retarded Primary and Secondary Growth in <i>Populus trichocarpa</i>}, volume={25}, ISSN={["1422-0067"]}, DOI={10.3390/ijms25042205}, abstractNote={Primary and secondary growth of trees are needed for increments in plant height and stem diameter, respectively, affecting the production of woody biomass for applications in timber, pulp/paper, and related biomaterials. These two types of growth are believed to be both regulated by distinct transcription factor (TF)-mediated regulatory pathways. Notably, we identified PtrLBD39, a highly stem phloem-specific TF in Populus trichocarpa and found that the ectopic expression of PtrLBD39 in P. trichocarpa markedly retarded both primary and secondary growth. In these overexpressing plants, the RNA-seq, ChIP-seq, and weighted gene co-expression network analysis (WGCNA) revealed that PtrLBD39 directly or indirectly regulates TFs governing vascular tissue development, wood formation, hormonal signaling pathways, and enzymes responsible for wood components. This regulation led to growth inhibition, decreased fibrocyte secondary cell wall thickness, and reduced wood production. Therefore, our study indicates that, following ectopic expression in P. trichocarpa, PtrLBD39 functions as a repressor influencing both primary and secondary growth.}, number={4}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Yu, Jing and Gao, Boyuan and Li, Danning and Li, Shuang and Chiang, Vincent L. and Li, Wei and Zhou, Chenguang}, year={2024}, month={Feb} }
 @article{dai_zhai_lin_wang_meng_li_mao_gao_ma_zhang_et al._2023, title={Cell-type-specific PtrWOX4a and PtrVCS2 form a regulatory nexus with a histone modification system for stem cambium development in Populus trichocarpa}, ISSN={["2055-0278"]}, DOI={10.1038/s41477-022-01315-7}, abstractNote={Stem vascular cambium cells in forest trees produce wood for materials and energy. WOX4 affects the proliferation of such cells in Populus. Here we show that PtrWOX4a is the most highly expressed stem vascular-cambium-specific (VCS) gene in P. trichocarpa, and its expression is controlled by the product of the second most highly expressed VCS gene, PtrVCS2, encoding a zinc finger protein. PtrVCS2 binds to the PtrWOX4a promoter as part of a PtrWOX13a-PtrVCS2-PtrGCN5-1-PtrADA2b-3 protein tetramer. PtrVCS2 prevented the interaction between PtrGCN5-1 and PtrADA2b-3, resulting in H3K9, H3K14 and H3K27 hypoacetylation at the PtrWOX4a promoter, which led to fewer cambium cell layers. These effects on cambium cell proliferation were consistent across more than 20 sets of transgenic lines overexpressing individual genes, gene-edited mutants and RNA interference lines in P. trichocarpa. We propose that the tetramer-PtrWOX4a system may coordinate genetic and epigenetic regulation to maintain normal vascular cambium development for wood formation.}, journal={NATURE PLANTS}, author={Dai, Xiufang and Zhai, Rui and Lin, Jiaojiao and Wang, Zhifeng and Meng, Dekai and Li, Meng and Mao, Yuli and Gao, Boyuan and Ma, Hongyan and Zhang, Baofeng and et al.}, year={2023}, month={Jan} }
 @article{liu_wang_liu_wang_fan_liu_zhang_yang_wang_sederoff_et al._2023, title={Exceptionally high genetic variance of the doubled haploid (DH) population of poplar}, volume={5}, ISSN={["1993-0607"]}, DOI={10.1007/s11676-023-01612-7}, abstractNote={Abstract Doubled haploid (DH) plants have been widely used for breeding and biological research in crops. Populus spp. have been used as model woody plant species for biological research. However, the induction of DH poplar plants is onerous, and limited biological or breeding work has been carried out on DH individuals or populations. In this study, we provide an effective protocol for poplar haploid induction based on an anther culture method. A total of 96 whole DH plant lines were obtained using an F 1 hybrid of Populus simonii × P. nigra as a donor tree. The phenotypes of the DH population showed exceptionally high variance when compared to those of half-sib progeny of the donor tree. Each DH line displayed distinct features compared to those of the other DH lines or the donor tree. Additionally, some excellent homozygous lines have the potential to be model plants in genetic and breeding studies.}, journal={JOURNAL OF FORESTRY RESEARCH}, author={Liu, Caixia and Wang, Sui and Liu, Yi and Wang, Meng and Fan, Erqin and Liu, Chen and Zhang, Shikai and Yang, Chuanping and Wang, Junhui and Sederoff, Heike W. W. and et al.}, year={2023}, month={May} }
 @article{sulis_jiang_yang_marques_matthews_miller_lan_cofre-vega_liu_sun_et al._2023, title={Multiplex CRISPR editing of wood for sustainable fiber production}, volume={381}, ISSN={["1095-9203"]}, url={http://europepmc.org/abstract/med/37440632}, DOI={10.1126/science.add4514}, abstractNote={The domestication of forest trees for a more sustainable fiber bioeconomy has long been hindered by the complexity and plasticity of lignin, a biopolymer in wood that is recalcitrant to chemical and enzymatic degradation. Here, we show that multiplex CRISPR editing enables precise woody feedstock design for combinatorial improvement of lignin composition and wood properties. By assessing every possible combination of 69,123 multigenic editing strategies for 21 lignin biosynthesis genes, we deduced seven different genome editing strategies targeting the concurrent alteration of up to six genes and produced 174 edited poplar variants. CRISPR editing increased the wood carbohydrate-to-lignin ratio up to 228% that of wild type, leading to more-efficient fiber pulping. The edited wood alleviates a major fiber-production bottleneck regardless of changes in tree growth rate and could bring unprecedented operational efficiencies, bioeconomic opportunities, and environmental benefits. Description Editor’s summary Trees provide an important natural resource, but breeding for optimal wood properties is time consuming and hindered by the complexity of tree genetics and diversity. Sulis et al. show that CRISPR technologies can be readily deployed to enhance wood properties and augment the sustainability of forest trees (see the Perspective by Zuin Zeidler). The authors generated multiplexed genetic alterations modifying wood composition in poplar with more desirable traits for fiber pulping and lower carbon emissions. This work demonstrates that genome editing can be harnessed for breeding more efficient trees, which will provide timely opportunities for sustainable forestry and a more efficient bioeconomy. —DJ Gene editing enables precise woody-feedstock design to enhance fiber production efficiency and sustainability in poplar trees.}, number={6654}, journal={SCIENCE}, author={Sulis, Daniel B. and Jiang, Xiao and Yang, Chenmin and Marques, Barbara M. and Matthews, Megan L. and Miller, Zachary and Lan, Kai and Cofre-Vega, Carlos and Liu, Baoguang and Sun, Runkun and et al.}, year={2023}, month={Jul}, pages={216-+} }
 @article{liu_gao_sun_li_zhang_wang_zhou_sulis_wang_chiang_et al._2022, title={Dimerization of PtrMYB074 and PtrWRKY19 mediates transcriptional activation of PtrbHLH186 for secondary xylem development in Populus trichocarpa}, volume={5}, ISSN={["1469-8137"]}, url={http://europepmc.org/abstract/med/35152419}, DOI={10.1111/nph.18028}, abstractNote={Summary Wood formation is controlled by transcriptional regulatory networks (TRNs) involving regulatory homeostasis determined by combinations of transcription factor (TF)–DNA and TF–TF interactions. Functions of TF–TF interactions in wood formation are still in the early stages of identification. PtrMYB074 is a woody dicot‐specific TF in a TRN for wood formation in Populus trichocarpa. Here, using yeast two‐hybrid and bimolecular fluorescence complementation, we conducted a genome‐wide screening for PtrMYB074 interactors and identified 54 PtrMYB074‐TF pairs. Of these pairs, 53 are novel. We focused on the PtrMYB074‐PtrWRKY19 pair, the most highly expressed and xylem‐specific interactor, and its direct transregulatory target, PtrbHLH186, the xylem‐specific one of the pair's only two direct TF target genes. Using transient and CRISPR‐mediated transgenesis in P. trichocarpa coupled with chromatin immunoprecipitation and electrophoretic mobility shift assays, we demonstrated that PtrMYB074 is recruited by PtrWRKY19 and that the PtrMYB074‐PtrWRKY19 dimers are required to transactive PtrbHLH186. Overexpressing PtrbHLH186 in P. trichocarpa resulted in retarded plant growth, increased guaiacyl lignin, a higher proportion of smaller stem vessels and strong drought‐tolerant phenotypes. Knowledge of the PtrMYB074‐PtrWRKY19‐PtrbHLH186 regulation may help design genetic controls of optimal growth and wood formation to maximize beneficial wood properties while minimizing negative effects on growth.}, journal={NEW PHYTOLOGIST}, author={Liu, Huizi and Gao, Jinghui and Sun, Jiatong and Li, Shuang and Zhang, Baofeng and Wang, Zhuwen and Zhou, Chenguang and Sulis, Daniel Barletta and Wang, Jack P. and Chiang, Vincent L. and et al.}, year={2022}, month={Mar} }
 @article{li_cai_zhang_pei_chen_jiang_han_zhao_li_zhang_et al._2022, title={The Manchurian Walnut Genome: Insights into Juglone and Lipid Biosynthesis}, volume={11}, ISSN={["2047-217X"]}, DOI={10.1093/gigascience/giac057}, abstractNote={Abstract Background Manchurian walnut (Juglans mandshurica Maxim.) is a tree with multiple industrial uses and medicinal properties in the Juglandaceae family (walnuts and hickories). J. mandshurica produces juglone, which is a toxic allelopathic agent and has potential utilization value. Furthermore, the seed of J. mandshurica is rich in various unsaturated fatty acids and has high nutritive value. Findings Here, we present a high-quality chromosome-scale reference genome assembly and annotation for J. mandshurica (n = 16) with a contig N50 of 21.4 Mb by combining PacBio high-fidelity reads with high-throughput chromosome conformation capture data. The assembled genome has an estimated sequence size of 548.7 Mb and consists of 657 contigs, 623 scaffolds, and 40,453 protein-coding genes. In total, 60.99% of the assembled genome consists of repetitive sequences. Sixteen super-scaffolds corresponding to the 16 chromosomes were assembled, with a scaffold N50 length of 33.7 Mb and a BUSCO complete gene percentage of 98.3%. J. mandshurica displays a close sequence relationship with Juglans cathayensis, with a divergence time of 13.8 million years ago. Combining the high-quality genome, transcriptome, and metabolomics data, we constructed a gene-to-metabolite network and identified 566 core and conserved differentially expressed genes, which may be involved in juglone biosynthesis. Five CYP450 genes were found that may contribute to juglone accumulation. NAC, bZip, NF-YA, and NF-YC are positively correlated with the juglone content. Some candidate regulators (e.g., FUS3, ABI3, LEC2, and WRI1 transcription factors) involved in the regulation of lipid biosynthesis were also identified. Conclusions Our genomic data provide new insights into the evolution of the walnut genome and create a new platform for accelerating molecular breeding and improving the comprehensive utilization of these economically important tree species.}, journal={GIGASCIENCE}, author={Li, Xiang and Cai, Kewei and Zhang, Qinhui and Pei, Xiaona and Chen, Song and Jiang, Luping and Han, Zhiming and Zhao, Minghui and Li, Yan and Zhang, Xinxin and et al.}, year={2022} }
 @article{yu_zhou_li_li_lin_wang_chiang_li_2022, title={p A PtrLBD39-mediated transcriptional network regulates tension wood formation in Populus trichocarpa}, volume={3}, ISSN={["2590-3462"]}, url={http://europepmc.org/abstract/med/35059630}, DOI={10.1016/j.xplc.2021.100250}, abstractNote={Tension wood (TW) is a specialized xylem tissue formed in angiosperm trees under gravitational stimulus or mechanical stresses (e.g., bending). The genetic regulation that underlies this important mechanism remains poorly understood. Here, we used laser capture microdissection of stem xylem cells coupled with full transcriptome RNA-sequencing to analyze TW formation in Populus trichocarpa. After tree bending, PtrLBD39 was the most significantly induced transcription factor gene; it has a phylogenetically paired homolog, PtrLBD22. CRISPR-based knockout of PtrLBD39/22 severely inhibited TW formation, reducing cellulose and increasing lignin content. Transcriptomic analyses of CRISPR-based PtrLBD39/22 double mutants showed that these two genes regulate a set of TW-related genes. Chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify direct targets of PtrLBD39. We integrated transcriptomic analyses and ChIP-seq assays to construct a transcriptional regulatory network (TRN) mediated by PtrLBD39. In this TRN, PtrLBD39 directly regulates 26 novel TW-responsive transcription factor genes. Our work suggests that PtrLBD39 and PtrLBD22 specifically control TW formation by mediating a TW-specific TRN in Populus.}, number={1}, journal={PLANT COMMUNICATIONS}, author={Yu, Jing and Zhou, Chenguang and Li, Danning and Li, Shuang and Lin, Ying-Chung Jimmy and Wang, Jack P. and Chiang, Vincent L. and Li, Wei}, year={2022}, month={Jan} }
 @article{matthews_wang_sederoff_chiang_williams_2021, title={A multiscale model of lignin biosynthesis for predicting bioenergy traits in Populus trichocarpa}, volume={19}, ISSN={["2001-0370"]}, url={http://europepmc.org/abstract/med/33425249}, DOI={10.1016/j.csbj.2020.11.046}, abstractNote={Understanding the mechanisms behind lignin formation is an important research area with significant implications for the bioenergy and biomaterial industries. Computational models are indispensable tools for understanding this complex process. Models of the monolignol pathway in Populus trichocarpa and other plants have been developed to explore how transgenic modifications affect important bioenergy traits. Many of these models, however, only capture one level of biological organization and are unable to capture regulation across multiple biological scales. This limits their ability to predict how gene modification strategies will impact lignin and other wood properties. While the first multiscale model of lignin biosynthesis in P. trichocarpa spanned the transcript, protein, metabolic, and phenotypic layers, it did not account for cross-regulatory influences that could impact abundances of untargeted monolignol transcripts and proteins. Here, we present a multiscale model incorporating these cross-regulatory influences for predicting lignin and wood traits from transgenic knockdowns of the monolignol genes. The three main components of this multiscale model are (1) a transcript-protein model capturing cross-regulatory influences, (2) a kinetic-based metabolic model, and (3) random forest models relating the steady state metabolic fluxes to 25 physical traits. We demonstrate that including the cross-regulatory behavior results in smaller predictive error for 23 of the 25 traits. We use this multiscale model to explore the predicted impact of novel combinatorial knockdowns on key bioenergy traits, and identify the perturbation of PtrC3H3 and PtrCAld5H1&2 monolignol genes as a candidate strategy for increasing saccharification efficiencies while reducing negative impacts on wood density and height.}, journal={COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL}, author={Matthews, Megan L. and Wang, Jack P. and Sederoff, Ronald and Chiang, Vincent L. and Williams, Cranos M.}, year={2021}, pages={168–182} }
 @misc{li_li_zhao_pang_wei_tigabu_chiang_sederoff_sederoff_zhao_2021, title={An Overview of the Practices and Management Methods for Enhancing Seed Production in Conifer Plantations for Commercial Use}, volume={7}, ISSN={["2311-7524"]}, DOI={10.3390/horticulturae7080252}, abstractNote={Flowering, the beginning of the reproductive growth, is a significant stage in the growth and development of plants. Conifers are economically and ecologically important, characterized by straight trunks and a good wood quality and, thus, conifer plantations are widely distributed around the world. In addition, conifer species have a good tolerance to biotic and abiotic stress, and a stronger survival ability. Seeds of some conifer species, such as Pinus koraiensis, are rich in vitamins, amino acids, mineral elements and other nutrients, which are used for food and medicine. Although conifers are the largest (giant sequoia) and oldest living plants (bristlecone pine), their growth cycle is relatively long, and the seed yield is unstable. In the present work, we reviewed selected literature and provide a comprehensive overview on the most influential factors and on the methods and techniques that can be adopted in order to improve flowering and seed production in conifers species. The review revealed that flowering and seed yields in conifers are affected by a variety of factors, such as pollen, temperature, light, water availability, nutrients, etc., and a number of management techniques, including topping off, pruning, fertilization, hormone treatment, supplementary pollination, etc. has been developed for improving cone yields. Furthermore, several flowering-related genes (FT, Flowering locus T and MADS-box, MCMI, AGAMOUS, DEFICIENCES and SRF) that play a crucial role in flowering in coniferous trees were identified. The results of this study can be useful for forest managers and for enhancing seed yields in conifer plantations for commercial use.}, number={8}, journal={HORTICULTURAE}, author={Li, Yan and Li, Xiang and Zhao, Ming-Hui and Pang, Zhong-Yi and Wei, Jia-Tong and Tigabu, Mulualem and Chiang, Vincent L. and Sederoff, Heike and Sederoff, Ronald and Zhao, Xi-Yang}, year={2021}, month={Aug} }
 @article{vries_brouckaert_chanoca_kim_regner_timokhin_sun_de meester_van doorsselaere_goeminne_et al._2021, title={CRISPR-Cas9 editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 shows their importance and partial redundancy in lignification in Populus tremula x P. alba}, volume={11}, ISSN={["1467-7652"]}, url={http://europepmc.org/abstract/med/34160888}, DOI={10.1111/pbi.13651}, abstractNote={Summary Lignins are cell wall‐located aromatic polymers that provide strength and hydrophobicity to woody tissues. Lignin monomers are synthesized via the phenylpropanoid pathway, wherein CAFFEOYL SHIKIMATE ESTERASE (CSE) converts caffeoyl shikimate into caffeic acid. Here, we explored the role of the two CSE homologs in poplar (Populus tremula × P. alba). Reporter lines showed that the expression conferred by both CSE1 and CSE2 promoters is similar. CRISPR‐Cas9‐generated cse1 and cse2 single mutants had a wild‐type lignin level. Nevertheless, CSE1 and CSE2 are not completely redundant, as both single mutants accumulated caffeoyl shikimate. In contrast, the cse1 cse2 double mutants had a 35% reduction in lignin and associated growth penalty. The reduced‐lignin content translated into a fourfold increase in cellulose‐to‐glucose conversion upon limited saccharification. Phenolic profiling of the double mutants revealed large metabolic shifts, including an accumulation of p‐coumaroyl, 5‐hydroxyferuloyl, feruloyl and sinapoyl shikimate, in addition to caffeoyl shikimate. This indicates that the CSEs have a broad substrate specificity, which was confirmed by in vitro enzyme kinetics. Taken together, our results suggest an alternative path within the phenylpropanoid pathway at the level of the hydroxycinnamoyl‐shikimates, and show that CSE is a promising target to improve plants for the biorefinery.}, journal={PLANT BIOTECHNOLOGY JOURNAL}, author={Vries, Lisanne and Brouckaert, Marlies and Chanoca, Alexandra and Kim, Hoon and Regner, Matthew R. and Timokhin, Vitaliy I. and Sun, Yi and De Meester, Barbara and Van Doorsselaere, Jan and Goeminne, Geert and et al.}, year={2021}, month={Aug} }
 @misc{zhang_yang_li_chiang_fu_2021, title={Cooperative Regulation of Flavonoid and Lignin Biosynthesis in Plants}, volume={40}, ISSN={["1549-7836"]}, DOI={10.1080/07352689.2021.1898083}, abstractNote={Abstract Flavonoids and lignin are valuable phytochemicals derived from plant secondary metabolism and play important roles in regulating multiple plant developmental processes and signaling networks. The biosynthetic pathways leading to flavonoids and lignin are known to be originated from the general phenylpropanoid pathway. Key regulators controlling the pathway structural genes have been isolated from many species. However, cooperative regulations of flavonoid and lignin biosynthesis and the resulting effects on the carbon flow in the general phenylpropanoid pathway have not systematically summarized and discussed. New discoveries have begun to reveal that the biosynthesis of flavonoids and lignin are linked through transcription regulatory networks sharing certain specific regulators, such as transcription factors, mediators and microRNAs. This review article summarizes recent progress on function and mechanism of these regulators and assesses how they co-modulate the biosynthesis of flavonoids and lignin. A simplified discussion for the different co-regulation networks involved with flavonoid and lignin biosynthesis is proposed.}, number={2}, journal={CRITICAL REVIEWS IN PLANT SCIENCES}, author={Zhang, Su and Yang, Jie and Li, Hongquan and Chiang, Vincent L. and Fu, Yujie}, year={2021}, month={Mar}, pages={109–126} }
 @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. In Populus 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 of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa. The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX 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 in P. 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} }
 @misc{li_he_gao_zhou_chiang_li_2021, title={Histone Acetylation Changes in Plant Response to Drought Stress}, volume={12}, ISSN={["2073-4425"]}, DOI={10.3390/genes12091409}, abstractNote={Drought stress causes recurrent damage to a healthy ecosystem because it has major adverse effects on the growth and productivity of plants. However, plants have developed drought avoidance and resilience for survival through many strategies, such as increasing water absorption and conduction, reducing water loss and conversing growth stages. Understanding how plants respond and regulate drought stress would be important for creating and breeding better plants to help maintain a sound ecosystem. Epigenetic marks are a group of regulators affecting drought response and resilience in plants through modification of chromatin structure to control the transcription of pertinent genes. Histone acetylation is an ubiquitous epigenetic mark. The level of histone acetylation, which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), determines whether the chromatin is open or closed, thereby controlling access of DNA-binding proteins for transcriptional activation. In this review, we summarize histone acetylation changes in plant response to drought stress, and review the functions of HATs and HDACs in drought response and resistance.}, number={9}, journal={GENES}, author={Li, Shuang and He, Xu and Gao, Yuan and Zhou, Chenguang and Chiang, Vincent L. and Li, Wei}, year={2021}, month={Sep} }
 @misc{yan_pei_zhang_li_zhang_zhao_chiang_sederoff_zhao_2021, title={MYB-Mediated Regulation of Anthocyanin Biosynthesis}, volume={22}, ISSN={["1422-0067"]}, DOI={10.3390/ijms22063103}, abstractNote={Anthocyanins are natural water-soluble pigments that are important in plants because they endow a variety of colors to vegetative tissues and reproductive plant organs, mainly ranging from red to purple and blue. The colors regulated by anthocyanins give plants different visual effects through different biosynthetic pathways that provide pigmentation for flowers, fruits and seeds to attract pollinators and seed dispersers. The biosynthesis of anthocyanins is genetically determined by structural and regulatory genes. MYB (v-myb avian myeloblastosis viral oncogene homolog) proteins are important transcriptional regulators that play important roles in the regulation of plant secondary metabolism. MYB transcription factors (TFs) occupy a dominant position in the regulatory network of anthocyanin biosynthesis. The TF conserved binding motifs can be combined with other TFs to regulate the enrichment and sedimentation of anthocyanins. In this study, the regulation of anthocyanin biosynthetic mechanisms of MYB-TFs are discussed. The role of the environment in the control of the anthocyanin biosynthesis network is summarized, the complex formation of anthocyanins and the mechanism of environment-induced anthocyanin synthesis are analyzed. Some prospects for MYB-TF to modulate the comprehensive regulation of anthocyanins are put forward, to provide a more relevant basis for further research in this field, and to guide the directed genetic modification of anthocyanins for the improvement of crops for food quality, nutrition and human health.}, number={6}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Yan, Huiling and Pei, Xiaona and Zhang, Heng and Li, Xiang and Zhang, Xinxin and Zhao, Minghui and Chiang, Vincent L. and Sederoff, Ronald Ross and Zhao, Xiyang}, year={2021}, month={Mar} }
 @article{li_li_zhao_hu_meng_song_tigabu_chiang_sederoff_ma_et al._2021, title={Molecular and Metabolic Insights into Anthocyanin Biosynthesis for Leaf Color Change in Chokecherry (Padus virginiana)}, volume={22}, ISSN={["1422-0067"]}, DOI={10.3390/ijms221910697}, abstractNote={Chokecherry (Padus virginiana L.) is an important landscaping tree with high ornamental value because of its colorful purplish-red leaves (PRL). The quantifications of anthocyanins and the mechanisms of leaf color change in this species remain unknown. The potential biosynthetic and regulatory mechanisms and the accumulation patterns of anthocyanins in P. virginiana that determine three leaf colors were investigated by combined analysis of the transcriptome and the metabolome. The difference of chlorophyll, carotenoid and anthocyanin content correlated with the formation of P. virginiana leaf color. Using enrichment and correlation network analysis, we found that anthocyanin accumulation differed in different colored leaves and that the accumulation of malvidin 3-O-glucoside (violet) and pelargonidin 3-O-glucoside (orange-red) significantly correlated with the leaf color change from green to purple-red. The flavonoid biosynthesis genes (PAL, CHS and CHI) and their transcriptional regulators (MYB, HD-Zip and bHLH) exhibited specific increased expression during the purple-red periods. Two genes encoding enzymes in the anthocyanin biosynthetic pathway, UDP glucose-flavonoid 3-O-glucosyl-transferase (UFGT) and anthocyanidin 3-O-glucosyltransferase (BZ1), seem to be critical for suppressing the formation of the aforesaid anthocyanins. In PRL, the expression of the genes encoding for UGFT and BZ1 enzymes was substantially higher than in leaves of other colors and may be related with the purple-red color change. These results may facilitate genetic modification or selection for further improvement in ornamental qualities of P. virginiana.}, number={19}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Li, Xiang and Li, Yan and Zhao, Minghui and Hu, Yanbo and Meng, Fanjuan and Song, Xingshun and Tigabu, Mulualem and Chiang, Vincent L. and Sederoff, Ronald and Ma, Wenjun and et al.}, year={2021}, month={Oct} }
 @article{liu_li_wang_fan_yang_wang_fu_ge_sederoff_sederoff_et al._2021, title={Qu-2, a robust poplar suspension cell line for molecular biology}, volume={32}, ISSN={["1993-0607"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85097191965&partnerID=MN8TOARS}, DOI={10.1007/s11676-020-01266-9}, abstractNote={Abstract Populus spp. have long been used as model woody plant species for molecular biology research. However, tissues of poplar are often recalcitrant to experimental procedures for molecular studies. We generated a hormone autotrophic poplar suspension cell line from a hybrid of Populus alba × P. berolinensis ‘Yinzhong’, named Qu-2. Qu-2 cells are suitable as a model biological system for studying woody plants. Qu-2 cells have many advantages over suspension cell lines derived so far from any other woody plants. Qu-2 cells are very easy to cultivate and can grow on several common plant culture media without the addition of any plant hormone. They show exceptionally high growth rates, reaching an approximately 150-fold increase in biomass after one week of culturing. Another important unique characteristic of Qu-2 cells is that they can be cryopreserved and readily reactivated. Qu-2 cells are suitable for molecular manipulations such as protoplast production, transient transformation, and RNA-seq analysis. Therefore, Qu-2 cells have the great potential to be an excellent model cell line in tree molecular biological research, ranging from physiology to gene function. The Qu-2 cells will be made available to the plant community for research.}, number={2}, journal={JOURNAL OF FORESTRY RESEARCH}, author={Liu, Caixia and Li, Kailong and Wang, Meng and Fan, Erqin and Yang, Chuanping and Wang, Junhui and Fu, Pengyue and Ge, Xiaolan and Sederoff, Heike W. and Sederoff, Ronald R. and et al.}, year={2021}, month={Apr}, pages={733–740} }
 @article{liu_liu_yu_wang_sun_li_lin_chiang_li_wang_2021, title={Transcriptional reprogramming of xylem cell wall biosynthesis in tension wood}, volume={186}, ISSN={["1532-2548"]}, url={https://doi.org/10.1093/plphys/kiab038}, DOI={10.1093/plphys/kiab038}, abstractNote={Abstract Tension wood (TW) is a specialized xylem tissue developed under mechanical/tension stress in angiosperm trees. TW development involves transregulation of secondary cell wall genes, which leads to altered wood properties for stress adaptation. We induced TW in the stems of black cottonwood (Populus trichocarpa, Nisqually-1) and identified two significantly repressed transcription factor (TF) genes: class B3 heat-shock TF (HSFB3-1) and MYB092. Transcriptomic analysis and chromatin immunoprecipitation (ChIP) were used to identify direct TF–DNA interactions in P. trichocarpa xylem protoplasts overexpressing the TFs. This analysis established a transcriptional regulatory network in which PtrHSFB3-1 and PtrMYB092 directly activate 8 and 11 monolignol genes, respectively. The TF–DNA interactions were verified for their specificity and transactivator roles in 35 independent CRISPR-based biallelic mutants and overexpression transgenic lines of PtrHSFB3-1 and PtrMYB092 in P. trichocarpa. The gene-edited trees (mimicking the repressed PtrHSFB3-1 and PtrMYB092 under tension stress) have stem wood composition resembling that of TW during normal growth and under tension stress (i.e., low lignin and high cellulose), whereas the overexpressors showed an opposite effect (high lignin and low cellulose). Individual overexpression of the TFs impeded lignin reduction under tension stress and restored high levels of lignin biosynthesis in the TW. This study offers biological insights to further uncover how metabolism, growth, and stress adaptation are coordinately regulated in trees.}, number={1}, journal={PLANT PHYSIOLOGY}, publisher={Oxford University Press (OUP)}, author={Liu, Baoguang and Liu, Juan and Yu, Jing and Wang, Zhifeng and Sun, Yi and Li, Shuang and Lin, Ying-Chung Jimmy and Chiang, Vincent L. and Li, Wei and Wang, Jack P.}, year={2021}, month={May}, pages={250–269} }
 @article{edmunds_peralta_sharma-shivappa_kelley_chiang_miller_giles_sykes_deoppke_gjersing_et al._2020, title={Fungal Pretreatment and Enzymatic Hydrolysis of Genetically-modified Populus trichocarpa}, volume={15}, ISSN={["1930-2126"]}, DOI={10.15376/biores.15.3.6488-6505}, abstractNote={Fungal pretreatment of Populus trichocarpa wood genetically modified to reduce lignin and alter lignin chemistry is investigated for its effectiveness as an alternative to common pretreatment methods. The goal of this work is to improve biomass utilization for biofuel and biochemical applications by increasing sugar release. Sugar release after enzymatic hydrolysis was measured after various biomass pretreatments (including wood-rot fungus, hot water, and dilute acid). In the wildtype, and in constructs downregulated in PAL, 4CL, and C3H, the fungal pretreatment resulted in substantial improvements in sugar yields, up to 2.4-fold increase in glucose yield and 6-fold increase in xylose yield after enzymatic hydrolysis compared to the unpretreated control. However, the effects of fungal pretreatment were inconsistent, and in genetic lines down-regulated in 4CL, CCoAOMT, CAld5H, and C3H, fungal pretreatment yielded similar or decreased sugar release after enzymatic hydrolysis.}, number={3}, journal={BIORESOURCES}, author={Edmunds, Charles W. and Peralta, Perry and Sharma-Shivappa, Ratna R. and Kelley, Stephen S. and Chiang, Vincent L. and Miller, Zachary D. and Giles, Richard L. and Sykes, Robert W. and Deoppke, Crissa and Gjersing, Erica and et al.}, year={2020}, month={Aug}, pages={6488–6505} }
 @misc{niu_wang_yuan_sederoff_sederoff_chiang_borriss_2020, title={Microbial Interactions Within Multiple-Strain Biological Control Agents Impact Soil-Borne Plant Disease}, volume={11}, ISSN={["1664-302X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85094139988&partnerID=MN8TOARS}, DOI={10.3389/fmicb.2020.585404}, abstractNote={Major losses of crop yield and quality caused by soil-borne plant diseases have long threatened the ecology and economy of agriculture and forestry. Biological control using beneficial microorganisms has become more popular for management of soil-borne pathogens as an environmentally friendly method for protecting plants. Two major barriers limiting the disease-suppressive functions of biocontrol microbes are inadequate colonization of hosts and inefficient inhibition of soil-borne pathogen growth, due to biotic and abiotic factors acting in complex rhizosphere environments. Use of a consortium of microbial strains with disease inhibitory activity may improve the biocontrol efficacy of the disease-inhibiting microbes. The mechanisms of biological control are not fully understood. In this review, we focus on bacterial and fungal biocontrol agents to summarize the current state of the use of single strain and multi-strain biological control consortia in the management of soil-borne diseases. We discuss potential mechanisms used by microbial components to improve the disease suppressing efficacy. We emphasize the interaction-related factors to be considered when constructing multiple-strain biological control consortia and propose a workflow for assembling them by applying a reductionist synthetic community approach.}, journal={FRONTIERS IN MICROBIOLOGY}, author={Niu, Ben and Wang, Weixiong and Yuan, Zhibo and Sederoff, Ronald R. and Sederoff, Heike and Chiang, Vincent L. and Borriss, Rainer}, year={2020}, month={Oct} }
 @article{straub_bing_wang_chiang_adams_kelly_2020, title={Use of the lignocellulose-degrading bacterium Caldicellulosiruptor bescii to assess recalcitrance and conversion of wild-type and transgenic poplar}, volume={13}, ISSN={["1754-6834"]}, url={http://europepmc.org/abstract/med/32180826}, DOI={10.1186/s13068-020-01675-2}, abstractNote={Abstract}, number={1}, journal={BIOTECHNOLOGY FOR BIOFUELS}, author={Straub, Christopher T. and Bing, Ryan G. and Wang, Jack P. and Chiang, Vincent L. and Adams, Michael W. W. and Kelly, Robert M.}, year={2020}, month={Mar} }
 @article{yeh_wang_miao_ma_kao_hsu_yu_hung_lin_kuan_et al._2019, title={A novel synthetic-genetic-array-based yeast one-hybrid system for high discovery rate and short processing time}, volume={29}, ISSN={["1549-5469"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85071056042&partnerID=MN8TOARS}, DOI={10.1101/gr.245951.118}, abstractNote={Eukaryotic gene expression is often tightly regulated by interactions between transcription factors (TFs) and their DNA cis targets. Yeast one-hybrid (Y1H) is one of the most extensively used methods to discover these interactions. We developed a high-throughput meiosis-directed yeast one-hybrid system using the Magic Markers of the synthetic genetic array analysis. The system has a transcription factor–DNA interaction discovery rate twice as high as the conventional diploid-mating approach and a processing time nearly one-tenth of the haploid-transformation method. The system also offers the highest accuracy in identifying TF–DNA interactions that can be authenticated in vivo by chromatin immunoprecipitation. With these unique features, this meiosis-directed Y1H system is particularly suited for constructing novel and comprehensive genome-scale gene regulatory networks for various organisms.}, number={8}, journal={GENOME RESEARCH}, author={Yeh, Chung-Shu and Wang, Zhifeng and Miao, Fang and Ma, Hongyan and Kao, Chung-Ting and Hsu, Tzu-Shu and Yu, Jhong-He and Hung, Er-Tsi and Lin, Chia-Chang and Kuan, Chen-Yu and et al.}, year={2019}, month={Aug}, pages={1343–1351} }
 @article{miller_peralta_mitchell_kelley_chiang_pearson_rottmann_cunningham_peszlen_2019, title={ANATOMICAL, PHYSICAL, AND MECHANICAL PROPERTIES OF TRANSGENIC LOBLOLLY PINE (PINUS TAEDA L.) MODIFIED FOR INCREASED DENSITY}, volume={51}, ISSN={["0735-6161"]}, DOI={10.22382/wfs-2019-018}, abstractNote={Traditional breeding methods are often constrained by the reproductive cycles of tree species and the difficulty in achieving significant improvements to complex traits; therefore, genetic manipulation of complex traits such as wood properties has the potential to resolve those issues. The objectives of this study were to analyze MOE, MOR, and the physical and anatomical properties of 2- to 3-yr-old field-grown transgenic Pinus taeda trees modified for increased density. This investigation consisted of a total of 55 sample trees in two separate experiments. Transgenic trees from sets OX41 and OX55, modified for increased density using two variants of the same HAP5 gene, exhibited higher mechanical properties with smaller stem diameter and tracheid lumen diameter than their set of control trees. In addition, set OX55 exhibited increased cell wall thickness. In the second experiment, the transgenic group WVK249, modified for higher density using an unrelated MYB gene, exhibited similar diameter growth and increased cell wall thickness and lower lumen/cell wall ratios but no change in mechanical properties compared with its control.}, number={2}, journal={WOOD AND FIBER SCIENCE}, author={Miller, Z. D. and Peralta, P. N. and Mitchell, P. H. and Kelley, S. S. and Chiang, V. L. and Pearson, L. and Rottmann, W. H. and Cunningham, M. W. and Peszlen, I. M.}, year={2019}, month={Apr}, pages={173–182} }
 @article{miller_peralta_mitchell_chiang_kelley_edmunds_peszlen_2019, title={Anatomy and Chemistry of Populus trichocarpa with Genetically Modified Lignin Content}, volume={14}, ISSN={["1930-2126"]}, DOI={10.15376/biores.14.3.5729-5746}, abstractNote={Vessel and fiber properties, diameter growth, and chemical compositions were investigated for transgenic Populus trichocarpa (black cottonwood) trees harvested after six months of growth in a greenhouse. Genetic modifications were cinnamyl alcohol dehydrogenase (CAD), cinnamate 3-hydroxylase (C3H), or cinnamate 4-hydroxylase (C4H), which resulted in modified lignin composition or content, and changed the syringyl-to-guaiacyl ratio. Comparing the genetic groups to the wild-type as the control, trees with reduced lignin content showed different results for vessel and fiber properties measured. Genetic groups with reduced PtrC3H3 and PtrC4H1&2 (with lower lignin content and higher S/G ratio than the control) exhibited splitting perpendicular to the rays, yet had the same fiber lumen diameter and the same fiber cell wall thickness as the control. Changes in lignin structure from modifications to PtrCAD resulted in reductions to the number of vessels, increases in vessel and fiber diameters, and had no consistent impact on stem diameter.}, number={3}, journal={BIORESOURCES}, author={Miller, Zachary D. and Peralta, Perry N. and Mitchell, Phil and Chiang, Vincent L. and Kelley, Stephen S. and Edmunds, Charles W. and Peszlen, Ilona M.}, year={2019}, month={Aug}, pages={5729–5746} }
 @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} }
 @misc{strauss_boerjan_chiang_costanza_coleman_davis_lu_mansfield_merkle_myburg_et al._2019, title={Certification for gene-edited forests}, volume={365}, ISSN={["1095-9203"]}, DOI={10.1126/science.aay6165}, number={6455}, journal={SCIENCE}, author={Strauss, Steven H. and Boerjan, Wout and Chiang, Vincent and Costanza, Adam and Coleman, Heather and Davis, John M. and Lu, Meng-Zhu and Mansfield, Shawn D. and Merkle, Scott and Myburg, Alexander and et al.}, year={2019}, month={Aug}, pages={767–768} }
 @misc{wang_liu_sun_chiang_sederoff_2019, title={Enzyme-Enzyme Interactions in Monolignol Biosynthesis}, volume={9}, ISSN={["1664-462X"]}, url={http://dx.doi.org/10.3389/fpls.2018.01942}, DOI={10.3389/fpls.2018.01942}, abstractNote={The enzymes that comprise the monolignol biosynthetic pathway have been studied intensively for more than half a century. A major interest has been the role of pathway in the biosynthesis of lignin and the role of lignin in the formation of wood. The pathway has been typically conceived as linear steps that convert phenylalanine into three major monolignols or as a network of enzymes in a metabolic grid. Potential interactions of enzymes have been investigated to test models of metabolic channeling or for higher order interactions. Evidence for enzymatic or physical interactions has been fragmentary and limited to a few enzymes studied in different species. Only recently the entire pathway has been studied comprehensively in any single plant species. Support for interactions comes from new studies of enzyme activity, co-immunoprecipitation, chemical crosslinking, bimolecular fluorescence complementation, yeast 2-hybrid functional screening, and cell type–specific gene expression based on light amplification by stimulated emission of radiation capture microdissection. The most extensive experiments have been done on differentiating xylem of Populus trichocarpa, where genomic, biochemical, chemical, and cellular experiments have been carried out. Interactions affect the rate, direction, and specificity of both 3 and 4-hydroxylation in the monolignol biosynthetic pathway. Three monolignol P450 mono-oxygenases form heterodimeric and heterotetrameric protein complexes that activate specific hydroxylation of cinnamic acid derivatives. Other interactions include regulatory kinetic control of 4-coumarate CoA ligases through subunit specificity and interactions between a cinnamyl alcohol dehydrogenase and a cinnamoyl-CoA reductase. Monolignol enzyme interactions with other pathway proteins have been associated with biotic and abiotic stress response. Evidence challenging or supporting metabolic channeling in this pathway will be discussed.}, journal={FRONTIERS IN PLANT SCIENCE}, author={Wang, Jack P. and Liu, Baoguang and Sun, Yi and Chiang, Vincent L. and Sederoff, Ronald R.}, year={2019}, month={Jan} }
 @article{wang_matthews_naik_williams_ducoste_sederoff_chiang_2019, title={Flux modeling for monolignol biosynthesis}, volume={56}, ISSN={0958-1669}, url={http://dx.doi.org/10.1016/J.COPBIO.2018.12.003}, DOI={10.1016/J.COPBIO.2018.12.003}, abstractNote={The pathway of monolignol biosynthesis involves many components interacting in a metabolic grid to regulate the supply and ratios of monolignols for lignification. The complexity of the pathway challenges any intuitive prediction of the output without mathematical modeling. Several models have been presented to quantify the metabolic flux for monolignol biosynthesis and the regulation of lignin content, composition, and structure in plant cell walls. Constraint-based models using data from transgenic plants were formulated to describe steady-state flux distribution in the pathway. Kinetic-based models using enzyme reaction and inhibition constants were developed to predict flux dynamics for monolignol biosynthesis in wood-forming cells. This review summarizes the recent progress in flux modeling and its application to lignin engineering for improved plant development and utilization.}, journal={Current Opinion in Biotechnology}, publisher={Elsevier BV}, author={Wang, Jack P and Matthews, Megan L and Naik, Punith P and Williams, Cranos M and Ducoste, Joel J and Sederoff, Ronald R and Chiang, Vincent L}, year={2019}, month={Apr}, pages={187–192} }
 @article{straub_khatibi_wang_conway_williams-rhaesa_peszlen_chiang_adams_kelly_2019, title={Quantitative fermentation of unpretreated transgenic poplar by Caldicellulosiruptor bescii}, volume={10}, ISSN={["2041-1723"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85070390326&partnerID=MN8TOARS}, DOI={10.1038/s41467-019-11376-6}, abstractNote={Abstract}, number={1}, journal={NATURE COMMUNICATIONS}, publisher={Springer Science and Business Media LLC}, author={Straub, Christopher T. and Khatibi, Piyum A. and Wang, Jack P. and Conway, Jonathan M. and Williams-Rhaesa, Amanda M. and Peszlen, Ilona M. and Chiang, Vincent L. and Adams, Michael W. W. and Kelly, Robert M.}, year={2019}, month={Aug} }
 @article{edmunds_peszlen_chiang_kelley_miller_davis_gjersing_peralta_2019, title={Thermo-mechanical Behavior of Genetically Modified Populus trichocarpa}, volume={14}, ISSN={["1930-2126"]}, DOI={10.15376/biores.14.2.4760-4773}, abstractNote={Wood processing is often performed at elevated temperatures under moisture-saturated conditions; therefore, it is important to understand the impact of the lignin content and lignin chemical structure on the thermo-mechanical properties of wood. In this study, genetically modified Populus trichocarpa wood specimens with down-regulated cinnamyl alcohol dehydrogenase, cinnamate 3-hydroxylase, and cinnamate 4-hydroxylase with altered lignin contents and/or lignin structures were utilized to probe the relationship between the lignin content, lignin monomer composition, and thermo-mechanical properties of solid wood. The thermo-mechanical properties of these unique samples were measured using dynamic mechanical analysis and the nuclear magnetic resonance (NMR) spin-spin relaxation time. The results showed that the transgenic P. trichocarpa samples had decreased storage and loss moduli compared with the wildtype. The solid-state NMR revealed increased lignin molecular mobility in the reduced-lignin transgenic lines. Also, noticeably reduced glass transition temperatures (Tg) were observed in the transgenic lines with reduced lignin contents and altered lignin monomer compositions compared with the wildtype. The increased lignin molecular mobility and reduced Tg in these samples can probably contribute to wood utilization and processing, such as lignin removal for pulp and paper and biofuels production, as well as particle consolidation during wood composite manufacturing.}, number={2}, journal={BIORESOURCES}, author={Edmunds, Charles W. and Peszlen, Ilona and Chiang, Vincent L. and Kelley, Stephen S. and Miller, Zachary D. and Davis, Mark F. and Gjersing, Erica and Peralta, Perry}, year={2019}, month={May}, pages={4760–4773} }
 @article{miller_peralta_mitchell_chiang_edmunds_peszlen_2018, title={Altered Lignin Content and Composition in Transgenic Populus trichocarpa Results in a Decrease of Modulus of Elasticity}, volume={13}, ISSN={["1930-2126"]}, DOI={10.15376/biores.13.4.7698-7708}, abstractNote={Transgenic Populus trichocarpa wood was compared to the corresponding wild-type material. The static modulus of elasticity in three-point bending was measured and the chemical composition among the specimens were compared, including the glucose, xylose, and lignin contents as well as the S/G ratio. Changes in chemical composition, created by genetic manipulations of the lignin biosynthetic pathway, affect the mechanical properties of young small-diameter transgenic trees. There are indications that a decrease in lignin content causes severe reductions in mechanical properties. Changes in lignin structure, either from an increased S/G ratio or structural lignin modifications, also negatively influence the mechanical properties.}, number={4}, journal={BIORESOURCES}, author={Miller, Zachary D. and Peralta, Perry N. and Mitchell, Phil and Chiang, Vincent L. and Edmunds, Charles W. and Peszlen, Ilona M.}, year={2018}, pages={7698–7708} }
 @article{naik_wang_sederoff_chiang_williams_ducoste_2018, title={Assessing the impact of the 4CL enzyme complex on the robustness of monolignol biosynthesis using metabolic pathway analysis}, volume={13}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0193896}, DOI={10.1371/journal.pone.0193896}, abstractNote={Lignin is a polymer present in the secondary cell walls of all vascular plants. It is a known barrier to pulping and the extraction of high-energy sugars from cellulosic biomass. The challenge faced with predicting outcomes of transgenic plants with reduced lignin is due in part to the presence of unique protein-protein interactions that influence the regulation and metabolic flux in the pathway. Yet, it is unclear why certain plants have evolved to create these protein complexes. In this study, we use mathematical models to investigate the role that the protein complex, formed specifically between Ptr4CL3 and Ptr4CL5 enzymes, have on the monolignol biosynthesis pathway. The role of this Ptr4CL3-Ptr4CL5 enzyme complex on the steady state flux distribution was quantified by performing Monte Carlo simulations. The effect of this complex on the robustness and the homeostatic properties of the pathway were identified by performing sensitivity and stability analyses, respectively. Results from these robustness and stability analyses suggest that the monolignol biosynthetic pathway is resilient to mild perturbations in the presence of the Ptr4CL3-Ptr4CL5 complex. Specifically, the presence of Ptr4CL3-Ptr4CL5 complex increased the stability of the pathway by 22%. The robustness in the pathway is maintained due to the presence of multiple enzyme isoforms as well as the presence of alternative pathways resulting from the presence of the Ptr4CL3-Ptr4CL5 complex.}, number={3}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Naik, Punith and Wang, Jack P. and Sederoff, Ronald and Chiang, Vincent and Williams, Cranos and Ducoste, Joel J.}, editor={Cullen, DanielEditor}, year={2018}, month={Mar}, pages={e0193896} }
 @article{wang_matthews_williams_shi_yang_tunlaya-anukit_chen_li_liu_lin_et al._2018, title={Improving wood properties for wood utilization through multi-omics integration in lignin biosynthesis}, volume={9}, ISSN={2041-1723}, url={http://dx.doi.org/10.1038/s41467-018-03863-z}, DOI={10.1038/s41467-018-03863-z}, abstractNote={Abstract}, number={1}, journal={Nature Communications}, publisher={Springer Science and Business Media LLC}, author={Wang, Jack P. and Matthews, Megan L. and Williams, Cranos M. and Shi, Rui and Yang, Chenmin and Tunlaya-Anukit, Sermsawat and Chen, Hsi-Chuan and Li, Quanzi and Liu, Jie and Lin, Chien-Yuan and et al.}, year={2018}, month={Apr}, pages={1579} }
 @article{edmunds_peralta_kelley_chiang_sharma-shivappa_davis_harman-ware_sykes_gjersing_cunningham_et al._2017, title={Characterization and enzymatic hydrolysis of wood from transgenic Pinus taeda engineered with syringyl lignin or reduced lignin content}, volume={24}, ISSN={["1572-882X"]}, DOI={10.1007/s10570-017-1231-z}, abstractNote={Softwood is an abundant resource; however, currently its utilization for bioconversion to obtain platform sugars is limited. Pinus taeda trees which were genetically modified to either produce S lignin or to decrease lignin content were characterized with a suite of analytic techniques. Syringyl lignin was visualized in the secondary xylem of one genetic line with Mäule staining. Solid-state nuclear magnetic resonance identified the S lignin units were coupled into the lignin through β-O-4 linkages, and thioacidolysis measured approximately 13% S lignin content in the same sample. Reductions of the lignin of as much as 33% were observed in the transgenics. To better understand how these modifications affect bioconversion, their amenability to hot water and dilute acid pretreatments and enzymatic hydrolysis was evaluated. Lignin reductions resulted in 1.9–3.2-fold increases in glucose release compared to the control. However, no apparent benefit was observed by S lignin incorporation at the concentrations reported in this study. These results highlight the potential for softwood cell wall properties to be improved for bioenergy/biochemical applications.}, number={4}, journal={CELLULOSE}, author={Edmunds, Charles W. and Peralta, Perry and Kelley, Stephen S. and Chiang, Vincent L. and Sharma-Shivappa, Ratna R. and Davis, Mark F. and Harman-Ware, Anne E. and Sykes, Robert W. and Gjersing, Erica and Cunningham, Michael W. and et al.}, year={2017}, month={Apr}, pages={1901–1914} }
 @article{xiang_sen_min_savithri_lu_jameel_chiang_chang_2017, title={Field-Grown Transgenic Hybrid Poplar with Modified Lignin Biosynthesis to Improve Enzymatic Saccharification Efficiency}, volume={5}, ISSN={["2168-0485"]}, DOI={10.1021/acssuschemeng.6b02740}, abstractNote={Hybrid poplars (Populus nigra L. × Populus maximowiczii A.) were genetically modified through antisense insertion of the 4-coumarate:coenzyme A ligase (4CL) gene. Compositional changes in response to this genetic change were measured in the field after 2 and 3 years of growth. The stem samples were treated with either green liquor or dilute acid pretreatments, representing alkaline and acid pretreatments. The enzymatic saccharification of the untreated and pretreated transgenic poplars were evaluated. After transgenic species were transplanted into the environment, they showed reduced recalcitrance to chemicals (i.e., pretreatments) and enzymes despite their lignin content and S/V ratio being comparable to those of the wild types. Compared to the field-grown poplars, the sugar yield increased up to 103% for untreated transgenic samples and increased 22% for acid- and green liquor-pretreated transgenic samples. This shows that field-grown transgenic hybrid poplars with modified lignin biosynthesis have imp...}, number={3}, journal={ACS SUSTAINABLE CHEMISTRY & ENGINEERING}, author={Xiang, Zhouyang and Sen, Suman Kumar and Min, Douyong and Savithri, Dhanalekshmi and Lu, Fachuang and Jameel, Hasan and Chiang, Vincent and Chang, Hou-min}, year={2017}, month={Mar}, pages={2407–2414} }
 @article{lin_chen_li_li_wang_shi_tunlaya-anukit_shuai_wang_ma_et al._2017, title={Reciprocal cross-regulation of VND and SND multigene TF families for wood formation in Populus trichocarpa}, volume={114}, ISSN={["0027-8424"]}, url={http://europepmc.org/abstract/med/29078399}, DOI={10.1073/pnas.1714422114}, abstractNote={Significance}, number={45}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Lin, Ying-Chung Jimmy and Chen, Hao and Li, Quanzi and Li, Wei and Wang, Jack P. and Shi, Rui and Tunlaya-Anukit, Sermsawat and Shuai, Peng and Wang, Zhifeng and Ma, Hongyan and et al.}, year={2017}, month={Nov}, pages={E9722–E9729} }
 @article{chang_sederoff_chiang_umezawa_2017, title={Takayoshi Higuchi (1927–2017)}, volume={37}, ISSN={0277-3813 1532-2319}, url={http://dx.doi.org/10.1080/02773813.2017.1350792}, DOI={10.1080/02773813.2017.1350792}, abstractNote={Takayoshi Higuchi, Professor Emeritus of Kyoto University, Japan, passed away from a heart attack at his home at the age of 89 on February 23, 2017. The wood science community has lost a great teacher, scientist, and true friend with his passing. He obtained his BSc degree at Nagoya University, Japan in 1950, majoring in biology and biochemistry, and ultimately obtained a Doctor of Agriculture from The University of Tokyo, Japan in 1959. Throughout his scientific career, he produced over 300 publications. From 1950 he worked at Gifu University as a Research Associate with Professor Ichiji Kawamura. At the same university, he was promoted to Lecturer in 1953, to Associate Professor in 1960, and to Professor in 1967. His research focused on lignin biodegradation by wood-rotting fungi and lignin biosynthesis in bamboo. In recognition of his biochemical studies of lignin formation, he was invited to the 4th International Congress of Biochemistry held in Vienna in 1958. There he became acquainted with several eminent professors working on lignin chemistry and biochemistry, including Professor Karl Freudenberg from Universität Heidelberg and Professor Karl Kratzl from Universität Wien, which made a lasting impression on his science. At that time, in the early 1960s, he went to Saskatoon, Canada, to work with Professor Stewart Brown. Shortly after leaving Canada, he worked with Professor Fernand Barnoud in Grenoble, France. While posted in Canada and France, Higuchi made several important contributions to monolignol biosynthesis through the establishment of key initial steps in the biosynthesis that made possible the development of our current knowledge of lignin formation. In 1968, Higuchi moved to Kyoto and was appointed Professor of Wood Research Institute, Kyoto University. He continued his biochemical and chemical studies of lignin biosynthesis, especially focusing on the role of O-methyltransferase in lignification, the characterization of p-hydroxyphenyl components of grass lignin, syringyl lignin formation, and preparation of a series of lignin model compounds that are indispensable in lignin reactivity studies. In the mid-1970s, Higuchi reinvigorated his lignin biodegradation studies, identifying specific reactions in lignin biodegradation and characterizing the reaction mechanisms of the lignin side chain and aromatic ring cleavages. From 1978 to 1984 and from 1988 to 1990, he was appointed Dean of the Wood Research Institute, Kyoto University. In 1970, he was elected as a fellow of the International Academy of Wood Science (IAWS). He was eventually elected to the Academy Board (1979–1983), Vice-president (1987– 1990), and President (1990–1993) of IAWS. In 1991, he was also appointed Foreign Associate of National Academy of Sciences, USA. Higuchi’s eminence in lignin biochemistry and chemistry was recognized by several awards: the Japan Prize of Agricultural Science (1985), the Anselme Payen Award of the American Chemical Society (1987), Dr. Honoris Causa from Grenoble University (1987), IAWS Academy Lecture (1988), the Medal with Purple Ribbon from the Government of Japan (1990), the Fujiwara Award (1992), the Order of the Sacred Treasure, Gold and Silver Star from the Government of Japan (2000), and the Japan Academy Prize (2001).}, number={5}, journal={Journal of Wood Chemistry and Technology}, publisher={Informa UK Limited}, author={Chang, Hou-min and Sederoff, Ron and Chiang, Vincent and Umezawa, Toshiaki}, year={2017}, month={Aug}, pages={405–406} }
 @article{shi_wang_lin_li_sun_chen_sederoff_chiang_2017, title={Tissue and cell-type co-expression networks of transcription factors and wood component genes in Populus trichocarpa}, volume={245}, ISSN={["1432-2048"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85009291513&partnerID=MN8TOARS}, DOI={10.1007/s00425-016-2640-1}, abstractNote={Co-expression networks based on transcriptomes of Populus trichocarpa major tissues and specific cell types suggest redundant control of cell wall component biosynthetic genes by transcription factors in wood formation. We analyzed the transcriptomes of five tissues (xylem, phloem, shoot, leaf, and root) and two wood forming cell types (fiber and vessel) of Populus trichocarpa to assemble gene co-expression subnetworks associated with wood formation. We identified 165 transcription factors (TFs) that showed xylem-, fiber-, and vessel-specific expression. Of these 165 TFs, 101 co-expressed (correlation coefficient, r > 0.7) with the 45 secondary cell wall cellulose, hemicellulose, and lignin biosynthetic genes. Each cell wall component gene co-expressed on average with 34 TFs, suggesting redundant control of the cell wall component gene expression. Co-expression analysis showed that the 101 TFs and the 45 cell wall component genes each has two distinct groups (groups 1 and 2), based on their co-expression patterns. The group 1 TFs (44 members) are predominantly xylem and fiber specific, and are all highly positively co-expressed with the group 1 cell wall component genes (30 members), suggesting their roles as major wood formation regulators. Group 1 TFs include a lateral organ boundary domain gene (LBD) that has the highest number of positively correlated cell wall component genes (36) and TFs (47). The group 2 TFs have 57 members, including 14 vessel-specific TFs, and are generally less correlated with the cell wall component genes. An exception is a vessel-specific basic helix-loop-helix (bHLH) gene that negatively correlates with 20 cell wall component genes, and may function as a key transcriptional suppressor. The co-expression networks revealed here suggest a well-structured transcriptional homeostasis for cell wall component biosynthesis during wood formation.}, number={5}, journal={PLANTA}, author={Shi, Rui and Wang, Jack P. and Lin, Ying-Chung and Li, Quanzi and Sun, Ying-Hsuan and Chen, Hao and Sederoff, Ronald R. and Chiang, Vincent L.}, year={2017}, month={May}, pages={927–938} }
 @article{lin_li_tunlaya-anukit_shi_sun_wang_liu_loziuk_edmunds_miller_et al._2016, title={A cell wall-bound anionic peroxidase, PtrPO21, is involved in lignin polymerization in Populus trichocarpa}, volume={12}, ISSN={1614-2942 1614-2950}, url={http://dx.doi.org/10.1007/S11295-016-0978-Y}, DOI={10.1007/s11295-016-0978-y}, number={2}, journal={Tree Genetics & Genomes}, publisher={Springer Science and Business Media LLC}, author={Lin, Chien-Yuan and Li, Quanzi and Tunlaya-Anukit, Sermsawat and Shi, Rui and Sun, Ying-Hsuan and Wang, Jack P. and Liu, Jie and Loziuk, Philip and Edmunds, Charles W. and Miller, Zachary D. and et al.}, year={2016}, month={Mar} }
 @article{kumari_deng_gunasekara_chiang_chen_ma_davis_wei_2016, title={Bottom-up GGM algorithm for constructing multilayered hierarchical gene regulatory networks that govern biological pathways or processes}, volume={17}, ISSN={["1471-2105"]}, DOI={10.1186/s12859-016-0981-1}, abstractNote={Abstract}, journal={BMC BIOINFORMATICS}, author={Kumari, Sapna and Deng, Wenping and Gunasekara, Chathura and Chiang, Vincent and Chen, Huann-sheng and Ma, Hao and Davis, Xin and Wei, Hairong}, year={2016}, month={Mar} }
 @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{loziuk_parker_li_lin_wang_li_sederoff_chiang_muddiman_2015, title={Elucidation of Xylem-Specific Transcription Factors and Absolute Quantification of Enzymes Regulating Cellulose Biosynthesis in Populus trichocarpa}, volume={14}, ISSN={["1535-3907"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84942916917&partnerID=MN8TOARS}, DOI={10.1021/acs.jproteome.5b00233}, abstractNote={Cellulose, the main chemical polymer of wood, is the most abundant polysaccharide in nature.1 The ability to perturb the abundance and structure of cellulose microfibrils is of critical importance to the pulp and paper industry as well as for the textile, wood products, and liquid biofuels industries. Although much has been learned at the transcript level about the biosynthesis of cellulose, a quantitative understanding at the proteome level has yet to be established. The study described herein sought to identify the proteins directly involved in cellulose biosynthesis during wood formation in Populus trichocarpa along with known xylem-specific transcription factors involved in regulating these key proteins. Development of an effective discovery proteomic strategy through a combination of subcellular fractionation of stem differentiating xylem tissue (SDX) with recently optimized FASP digestion protocols, StageTip fractionation, as well as optimized instrument parameters for global proteomic analysis using the quadrupole-orbitrap mass spectrometer resulted in the deepest proteomic coverage of SDX protein from P. trichocarpa with 9,146 protein groups being identified (1% FDR). Of these, 20 cellulosic/hemicellulosic enzymes and 43 xylem-specific transcription factor groups were identified. Finally, selection of surrogate peptides led to an assay for absolute quantification of 14 cellulosic proteins in SDX of P. trichocarpa.}, number={10}, journal={JOURNAL OF PROTEOME RESEARCH}, author={Loziuk, Philip L. and Parker, Jennifer and Li, Wei and Lin, Chien-Yuan and Wang, Jack P. and Li, Quanzi and Sederoff, Ronald R. and Chiang, Vincent L. and Muddiman, David C.}, year={2015}, month={Oct}, pages={4158–4168} }
 @article{wang_chuang_loziuk_chen_lin_shi_qu_muddiman_sederoff_chiang_2015, title={Phosphorylation is an on/off switch for 5-hydroxyconiferaldehyde O-methyltransferase activity in poplar monolignol biosynthesis}, volume={112}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/PNAS.1510473112}, DOI={10.1073/pnas.1510473112}, abstractNote={Significance}, number={27}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Wang, Jack P. and Chuang, Ling and Loziuk, Philip L. and Chen, Hao and Lin, Ying-Chung and Shi, Rui and Qu, Guan-Zheng and Muddiman, David C. and Sederoff, Ronald R. and Chiang, Vincent L.}, year={2015}, month={Jun}, pages={8481–8486} }
 @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{xiang_sen_roy_min_savithri_jameel_chiang_chang_2015, title={Wood characteristics and enzymatic saccharification efficiency of field-grown transgenic black cottonwood with altered lignin content and structure}, volume={22}, ISSN={["1572-882X"]}, DOI={10.1007/s10570-014-0541-7}, number={1}, journal={CELLULOSE}, author={Xiang, Zhouyang and Sen, Suman Kumar and Roy, Aparna and Min, Douyong and Savithri, Dhanalekshmi and Jameel, Hasan and Chiang, Vincent and Chang, Hou-min}, year={2015}, month={Feb}, pages={683–693} }
 @article{nakatsubo_ragamustari_hattori_ono_yamamura_li_chiang_umezawa_2014, title={A new O-methyltransferase for monolignol synthesis in Carthamus tinctorius}, volume={31}, ISSN={["1342-4580"]}, DOI={10.5511/plantbiotechnology.14.0903a}, abstractNote={A novel type of O-methyltransferase (OMT) cDNA was isolated from maturing seeds of Carthamus tinctorius (safflower). The deduced sequence of the OMT protein showed moderate sequence identity (52%) with C. tinctorius 5-hydroxyconiferaldehyde O-methyltransferase 1 (CAldOMT1). Phylogenetic analysis showed that the novel OMT did not belong to the typical CAldOMT [=caffeic acid OMT (CAOMT)] cluster. The recombinant protein of the OMT catalyzed 3(or 5-) O-methylation of hydroxycinnamaldehydes and hydroxycinnamyl alcohols, while it showed only weak or moderate activity toward hydroxycinnamates and hydroxycinnamoyl coenzyme A esters. Therefore, this OMT was designated as C. tinctorius 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol OMT (CtAAOMT). The time profile of CtAAOMT gene expression in C. tinctorius matched the patterns of lignin accumulation. Taken together, our data strongly suggest that along with CtCAldOMT1, CtAAOMT is involved in biosynthesis of syringyl lignin.}, number={5}, journal={Plant Biotechnology}, author={Nakatsubo, Tomoyuki and Ragamustari, Safendrri Komara and Hattori, Takefumi and Ono, Eiichiro and Yamamura, Masaomi and Li, Laigeng and Chiang, Vincent L. and Umezawa, Toshiaki}, year={2014}, month={Dec}, pages={545–553} }
 @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{loziuk_sederoff_chiang_muddiman_2014, title={Establishing ion ratio thresholds based on absolute peak area for absolute protein quantification using protein cleavage isotope dilution mass spectrometry}, volume={139}, ISSN={["1364-5528"]}, DOI={10.1039/c4an00567h}, abstractNote={Relative abundance values and their associated variability are dynamic and dependent on absolute abundance.}, number={21}, journal={ANALYST}, author={Loziuk, Philip L. and Sederoff, Ronald R. and Chiang, Vincent L. and Muddiman, David C.}, year={2014}, pages={5439–5450} }
 @article{min_xiang_liu_jameel_chiang_jin_chang_2014, title={Improved Protocol for Alkaline Nitrobenzene Oxidation of Woody and Non-Woody Biomass}, volume={35}, ISSN={0277-3813 1532-2319}, url={http://dx.doi.org/10.1080/02773813.2014.902965}, DOI={10.1080/02773813.2014.902965}, abstractNote={The protocol of alkaline nitrobenzene oxidation was investigated to improve its ability to identify the different lignin structures for both woody and non-woody biomass. The survival factors of all six oxidation products—syringaldehde (Sr), vanillin (V), p-hydroxybenzaldehyde (B) and their corresponding acids, syringic acid (Sa), vanillic acid (Va), and p-hydroxybenzoic acid (Ba)—were studied at 170, 180, and 190°C for several residence times. Under similar conditions, various lignin model compounds—a softwood (loblolly pine), a hardwood (red maple), and a non-wood raw material (corn stover)—were oxidized. Molar yields of oxidation products were determined and the ratios of (Sr+Sa)/(V+Va), (Sr/V), and B/(V+Va) (B/V) were calculated. All oxidation products were relatively stable at 170 and 180°C but showed some degradation at 190°C, especially at long residence time. In all cases, p-hydroxybenzoic acid was barely detectable. While yields of oxidation products reach a maximum at 170°C for pine and maple, maximal yields of corn stover require 190°C. Consequently, we recommend that nitrobenzene oxidation be carried out at 170°C for 2.5 h for softwood and hardwood, but at 190°C and 4 h with correction for the survival factors for corn stover and other non-woody biomass. Alternatively, a protocol of oxidation at two temperatures is recommended for non-woody biomass.}, number={1}, journal={Journal of Wood Chemistry and Technology}, publisher={Informa UK Limited}, author={Min, Douyong and Xiang, Zhouyang and Liu, Jie and Jameel, Hasan and Chiang, Vincent and Jin, Yongcan and Chang, Hou-min}, year={2014}, month={Sep}, pages={52–61} }
 @article{sutela_hahl_tiimonen_aronen_ylioja_laakso_saranpaa_chiang_julkunen-tiitto_haggman_2014, title={Phenolic Compounds and Expression of 4CL Genes in Silver Birch Clones and Pt4CL1a Lines}, volume={9}, ISSN={["1932-6203"]}, DOI={10.1371/journal.pone.0114434}, abstractNote={A small multigene family encodes 4-coumarate:CoA ligases (4CLs) catalyzing the CoA ligation of hydroxycinnamic acids, a branch point step directing metabolites to a flavonoid or monolignol pathway. In the present study, we examined the effect of antisense Populus tremuloides 4CL (Pt4CL1) to the lignin and soluble phenolic compound composition of silver birch (Betula pendula) Pt4CL1a lines in comparison with non-transgenic silver birch clones. The endogenous expression of silver birch 4CL genes was recorded in the stems and leaves and also in leaves that were mechanically injured. In one of the transgenic Pt4CL1a lines, the ratio of syringyl (S) and guaiacyl (G) lignin units was increased. Moreover, the transcript levels of putative silver birch 4CL gene (Bp4CL1) were reduced and contents of cinnamic acid derivatives altered. In the other two Pt4CL1a lines changes were detected in the level of individual phenolic compounds. However, considerable variation was found in the transcript levels of silver birch 4CLs as well as in the concentration of phenolic compounds among the transgenic lines and non-transgenic clones. Wounding induced the expression of Bp4CL1 and Bp4CL2 in leaves in all clones and transgenic lines, whereas the transcript levels of Bp4CL3 and Bp4CL4 remained unchanged. Moreover, minor changes were detected in the concentrations of phenolic compounds caused by wounding. As an overall trend the wounding decreased the flavonoid content in silver birches and increased the content of soluble condensed tannins. The results indicate that by reducing the Bp4CL1 transcript levels lignin composition could be modified. However, the alterations found among the Pt4CL1a lines and the non-transgenic clones were within the natural variation of silver birches, as shown in the present study by the clonal differences in the transcripts levels of 4CL genes, soluble phenolic compounds and condensed tannins.}, number={12}, journal={PLOS ONE}, author={Sutela, Suvi and Hahl, Terhi and Tiimonen, Heidi and Aronen, Tuija and Ylioja, Tiina and Laakso, Tapio and Saranpaa, Pekka and Chiang, Vincent and Julkunen-Tiitto, Riitta and Haggman, Hely}, year={2014}, month={Dec} }
 @article{li_gunasekara_guo_zhang_lei_tunlaya-anukit_busov_chiang_wei_2014, title={Pop's Pipes: poplar gene expression data analysis pipelines}, volume={10}, ISSN={["1614-2950"]}, DOI={10.1007/s11295-014-0745-x}, number={4}, journal={TREE GENETICS & GENOMES}, author={Li, Xiang and Gunasekara, Chathura and Guo, Yufeng and Zhang, Hang and Lei, Liang and Tunlaya-Anukit, Sermsawat and Busov, Victor and Chiang, Vincent and Wei, Hairong}, year={2014}, month={Aug}, pages={1093–1101} }
 @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_yang_chiang_jameel_chang_2014, title={The influence of lignin-carbohydrate complexes on the cellulase-mediated saccharification II: Transgenic hybrid poplars (Populus nigra L. and Populus maximowiczii A.)}, volume={116}, ISSN={["1873-7153"]}, DOI={10.1016/j.fuel.2013.07.046}, abstractNote={Twelve transgenic hybrid poplars (Populus nigra L. and Populus maximowiczii A.) were used to demonstrate the influence of the lignin–carbohydrate complexes (LCCs) on enzymatic saccharification. The samples have different levels of the syringaldehyde (S) to vanillin (V) ratio from 0.1 to 2.6 and the lignin content from 10.5% to 24.3%, compared to the control (the S/V ratio 1.7 and the lignin content 22.0%). Any adventitious contaminants that could affect the final enzymatic saccharification were removed by performing thorough extraction on the samples. The crude milled wood lignins were subsequently isolated from which the phenyl glycoside, benzyl ether and γ-ester linkages representative of the LCC were identified and quantified by 13C and 1H–13C HSQC NMR. It was found that the samples showed different levels of the three LCC linkages, depending on the lignin content and/or the S/V ratio. The correlation between the LCCs and enzymatic saccharification nearly conclusively demonstrated that the LCCs accounted for the recalcitrance of lignocellulosic biodegradation.}, journal={FUEL}, author={Min, Dou-yong and Yang, Chenmin and Chiang, Vincent and Jameel, Hasan and Chang, Hou-min}, year={2014}, month={Jan}, pages={56–62} }
 @article{kafle_shi_lee_mittal_park_sun_park_chiang_kim_2014, title={Vibrational sum-frequency-generation (SFG) spectroscopy study of the structural assembly of cellulose microfibrils in reaction woods}, volume={21}, ISSN={["1572-882X"]}, DOI={10.1007/s10570-014-0322-3}, number={4}, journal={CELLULOSE}, author={Kafle, Kabindra and Shi, Rui and Lee, Christopher M. and Mittal, Ashutosh and Park, Yong Bum and Sun, Ying-Hsuan and Park, Sunkyu and Chiang, Vincent and Kim, Seong H.}, year={2014}, month={Aug}, pages={2219–2231} }
 @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{umezawa_ragamustari_nakatsubo_wada_li_yamamura_sakakibara_hattori_suzuki_chiang_2013, title={A lignan O-methyltransferase catalyzing the regioselective methylation of matairesinol in Carthamus tinctorius}, volume={30}, ISSN={["1342-4580"]}, DOI={10.5511/plantbiotechnology.12.1230a}, abstractNote={Lignans are a group of plant phenolic compounds with various biological activities, including antitumor and antioxidant properties. O-Methylation is a critical step in biosynthesis of these compounds. However, little is known about the O-methyltransferase (OMT) enzymes that catalyze lignan O-methylation. We discovered a highly regioselective OMT activity in safflower (Carthamus tinctorius) seeds that catalyzed the methylation of matairesinol, a dibenzylbutyrolactone lignan, into 4′-O-methylmatairesinol (arctigenin) but not 4-O-methylmatairesinol (isoarctigenin). By examining such OMT activity in correlation with OMT transcript abundances during seed development, we cloned a few putative OMT cDNAs and produced their recombinant proteins in Escherichia coli. Among them, one protein exhibited O-methylation activity for matairesinol with the regioselectivity identical to that of the plant protein, and was named C. tinctorius matairesinol OMT (CtMROMT). CtMROMT did not show any detectable OMT activities towards phenylpropanoid monomers under the reaction conditions tested, while it methylated flavonoid apigenin efficiently into 4′-O-methylapigenin (acacetin). However, quantitative real-time polymerase chain reaction analysis demonstrated that expression of the CtMROMT gene was synchronized with the CtMROMT activity profile and arctigenin accumulation in the plant. These results demonstrated that CtMROMT is a novel plant OMT for lignan methylation.}, number={2}, journal={PLANT BIOTECHNOLOGY}, author={Umezawa, Toshiaki and Ragamustari, Safendrri Komara and Nakatsubo, Tomoyuki and Wada, Shohei and Li, Laigeng and Yamamura, Masaomi and Sakakibara, Norikazu and Hattori, Takefumi and Suzuki, Shiro and Chiang, Vincent L.}, year={2013}, pages={97–109} }
 @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{albert_barbazuk_depamphilis_der_leebens-mack_ma_palmer_rounsley_sankoff_schuster_et al._2013, title={The Amborella Genome and the Evolution of Flowering Plants}, volume={342}, ISSN={["1095-9203"]}, url={http://science.sciencemag.org/content/342/6165/1241089.long}, DOI={10.1126/science.1241089}, abstractNote={Shaping Plant Evolution}, number={6165}, journal={Science}, author={Albert, Victor A. and Barbazuk, W. Bradley and dePamphilis, Claude W. and Der, Joshua P. and Leebens-Mack, James and Ma, Hong and Palmer, Jeffrey D. and Rounsley, Steve and Sankoff, David and Schuster, Stephan C. and et al.}, year={2013}, month={Dec}, pages={1456–1457} }
 @article{min_yang_shi_jameel_chiang_chang_2013, title={The elucidation of the lignin structure effect on the cellulase-mediated saccharification by genetic engineering poplars (Populus nigra L. x Populus maximowiczii A.)}, volume={58}, ISSN={["1873-2909"]}, DOI={10.1016/j.biombioe.2013.08.019}, abstractNote={The lignin structure effect on the recalcitrance of biomass degradation was elucidated by twelve transgenic hybrid poplars (Populus nigra L. and Populus maximowiczii A.). Samples have different levels of the syringaldehyde (S) to vanillin (V) ratio from 0.1 to 2.6 and the lignin content from 10.5 to 24.3%, compared to the control (the S to V ratio 1.7 and the lignin content 22.0%). The dilute acid (0.1% sulfuric acid, 185 °C, and 30 min) and the green liquor (6% total titratable alkali (TTA), 25% sulfidity based on TTA, 185 °C and 15 min) were used as pretreatments. The enzymatic saccharification was performed at 5 FPU g−1 based on the substrate. The removal of lignin and hemicelluloses varied with pretreatments. The high delignification originated from the high S to V ratio. However, the high S to V ratio retarded the enzymatic saccharification. The decrease of the lignin and the hemicelluloses benefited the enzymatic saccharification with or without the pretreatment.}, journal={BIOMASS & BIOENERGY}, author={Min, Douyong and Yang, Chenmin and Shi, Rui and Jameel, Hasan and Chiang, Vincent and Chang, Houmin}, year={2013}, month={Nov}, pages={52–57} }
 @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{sun_shi_zhang_chiang_sederoff_2012, title={MicroRNAs in trees}, volume={80}, ISSN={["0167-4412"]}, DOI={10.1007/s11103-011-9864-z}, abstractNote={MicroRNAs (miRNAs) are 20-24 nucleotide long molecules processed from a specific class of RNA polymerase II transcripts that mainly regulate the stability of mRNAs containing a complementary sequence by targeted degradation in plants. Many features of tree biology are regulated by miRNAs affecting development, metabolism, adaptation and evolution. MiRNAs may be modified and harnessed for controlled suppression of specific genes to learn about gene function, or for practical applications through genetic engineering. Modified (artificial) miRNAs act as dominant suppressors and are particularly useful in tree genetics because they bypass the generations of inbreeding needed for fixation of recessive mutations. The purpose of this review is to summarize the current status of information on miRNAs in trees and to guide future studies on the role of miRNAs in the biology of woody perennials and to illustrate their utility in directed genetic modification of trees.}, number={1}, journal={PLANT MOLECULAR BIOLOGY}, author={Sun, Ying-Hsuan and Shi, Rui and Zhang, Xing-Hai and Chiang, Vincent L. and Sederoff, Ronald R.}, year={2012}, month={Sep}, pages={37–53} }
 @article{shuford_sederoff_chiang_muddiman_2012, title={Peptide Production and Decay Rates Affect the Quantitative Accuracy of Protein Cleavage Isotope Dilution Mass Spectrometry (PC-IDMS)}, volume={11}, ISSN={1535-9476 1535-9484}, url={http://dx.doi.org/10.1074/mcp.O112.017145}, DOI={10.1074/mcp.O112.017145}, abstractNote={No consensus has been reached on the proper time to add stable-isotope labeled (SIL) peptides in protein cleavage isotope dilution mass spectrometry workflows. While quantifying 24 monolignol pathway enzymes in the xylem tissue of Populus trichocarpa, we compared the protein concentrations obtained when adding the SIL standard peptides concurrently with the enzyme or after quenching of the digestion (i.e. postdigestion) and observed discrepancies for nearly all tryptic peptides investigated. In some cases, greater than 30-fold differences were observed. To explain these differences and potentially correct for them, we developed a mathematical model based on pseudo-first-order kinetics to account for the dynamic production and decay (e.g. degradation and precipitation) of the native peptide targets in conjunction with the decay of the SIL peptide standards. A time course study of the digests confirmed the results predicted by the proposed model and revealed that the discrepancy between concurrent and postdigestion introduction of the SIL standards was related to differential decay experienced by the SIL peptide and the native peptide in each method. Given these results, we propose concurrent introduction of the SIL peptide is most appropriate, though not free from bias. Mathematical modeling of this method reveals that overestimation of protein quantities would still result when rapid peptide decay occurs and that this bias would be further exaggerated by slow proteolysis. We derive a simple equation to estimate the bias for each peptide based on the relative rates of production and decay. According to this equation, nearly half of the peptides evaluated here were estimated to have quantitative errors greater than 10% and in a few cases over 100%. We conclude that the instability of peptides can often significantly bias the protein quantities measured in protein cleavage isotope dilution mass spectrometry-based assays and suggest peptide stability be made a priority when selecting peptides to use for quantification.}, number={9}, journal={Molecular & Cellular Proteomics}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Shuford, Christopher M. and Sederoff, Ronald R. and Chiang, Vincent L. and Muddiman, David C.}, year={2012}, month={May}, pages={814–823} }
 @article{perera_schmidt_chiang_schuck_adams_2012, title={Raman-spectroscopy-based noninvasive microanalysis of native lignin structure}, volume={402}, ISSN={["1618-2642"]}, DOI={10.1007/s00216-011-5518-x}, abstractNote={A new robust, noninvasive, Raman microspectroscopic method is introduced to analyze the structure of native lignin. Lignin spectra of poplar, Arabidopsis, and Miscanthus were recovered and structural differences were unambiguously detected. Compositional analysis of 4-coumarate-CoA ligase suppressed transgenic poplar showed that the syringyl-to-guaiacyl ratio decreased by 35% upon the mutation. A cell-specific compositional analysis of basal stems of Arabidopsis showed similar distributions of S and G monolignols in xylary fiber cells and interfascicular cells.}, number={2}, journal={ANALYTICAL AND BIOANALYTICAL CHEMISTRY}, author={Perera, Pradeep N. and Schmidt, Martin and Chiang, Vincent L. and Schuck, P. James and Adams, Paul D.}, year={2012}, month={Jan}, pages={983–987} }
 @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} }
 @inproceedings{min_li_yang_chang_chiang_jameel_2012, title={The effects of transgenic samples on the enzymatic saccharification}, booktitle={Proceeding of the 4th International Conference on Pulping, Papermaking and Biotechnology (ICPPB '12), vols. I and II}, author={Min, D. Y. and Li, Q. Z. and Yang, C. M. and Chang, H. M. and Chiang, V. and Jameel, H.}, year={2012}, pages={1058–1062} }
 @article{min_jameel_chiang_chang_2012, title={effect of lignin on enzymatic saccharification of hardwood after green liquor and sulfuric acid pretreatments}, volume={7}, DOI={10.15376/biores.7.2.2272-2283}, abstractNote={Red maple, sweet gum, trembling aspen, red alder, and Eucalyptus globulus samples were pretreated with dilute sulfuric acid and green liquor before enzymatic saccharification. Substrates showed different levels of delignification and sugar recovery, depending on the applied pretreatments and the syringaldehyde/vanillin ratio (S/V). Three major conclusions were drawn in this research. First, lignin is the greatest contributor to recalcitrance of hardwood to enzymatic saccharification. Second, a high S/V ratio is a useful indicator of high delignification during a pretreatment process. Third, green liquor pretreatment is a promising pretreatment method because of a high delignification degree and sugar recovery. In addition, xylan also contributes to the recalcitrance of hardwoods toward enzymatic saccharification.}, number={2}, journal={BioResources}, author={Min, D. Y. and Jameel, H. and Chiang, V. and Chang, H. M.}, year={2012}, pages={2272–2283} }
 @article{zhou_li_chiang_lucia_griffis_2011, title={Chemical and Spatial Differentiation of Syringyl and Guaiacyl Lignins in Poplar Wood via Time-of-Flight Secondary Ion Mass Spectrometry}, volume={83}, ISSN={["1520-6882"]}, DOI={10.1021/ac200903y}, abstractNote={As a major component in plant cell walls, lignin is an important factor in numerous industrial processes, especially in wood saccharification and fermentation to biofuels. The ability to chemically differentiate and spatially locate lignins in wood cell structures provides an important contribution to the effort to improve these processes. The spatial distribution of the syringyl (S) and guaiacyl (G) lignins, both over larger regions and within a single cell wall, on poplar ( Populus trichocarpa ) wood cross-sections was determined via time-of-flight secondary ion mass spectrometry (ToF-SIMS). This is the first time that direct chemically specific mass spectrometric mapping has been employed to elucidate the spatial distribution of S and G lignins. In agreement with results obtained by UV microscopy, ToF-SIMS images clearly show that the guaiacyl lignin is predominantly located in the vessel cell walls of poplar wood while syringyl lignin is mainly located in the fiber cell walls. The G/S ratio in vessel cell walls was determined to be approximately twice that found in fiber cell walls. A combination of Bi ToF-SIMS spectral image acquisition and C(60) sputtering provided the ability to attain the combination of spatial resolution and signal-to-noise necessary to determine the distribution of S and G lignins in a single cell wall. By this technique, it was possible to demonstrate that more guaiacyl lignin is located in the middle lamella layer and more syringyl lignin is located in the inner cell wall area.}, number={18}, journal={ANALYTICAL CHEMISTRY}, author={Zhou, Chuanzhen and Li, Quanzi and Chiang, Vincent L. and Lucia, Lucian A. and Griffis, Dieter P.}, year={2011}, month={Sep}, pages={7020–7026} }
 @article{min_li_jameel_chiang_chang_2011, title={Comparison of pretreatment protocols for cellulase-mediated saccharification of wood derived from transgenic low-xylan lines of cottonwood (P. trichocarpa)}, volume={35}, ISSN={["1873-2909"]}, DOI={10.1016/j.biombioe.2011.04.034}, abstractNote={The novel low xylan content transgenic cottonwood (P. trichocarpa) was used to elucidate recalcitrance of enzymatic saccharification with or without four different pretreatment methods. The xylan contents of two transgenic samples (8Di3 and 8Di5) were 11.4% and 11.7%, respectively, as compared with the wild type (16.0%). Contrarily, the lignin contents of two transgenic samples were 23.1% and 24.5%, respectively, as compared with the wild type (20.8%). The four pretreatments were dilute acid (0.1% sulfuric acid, 185 °C, 30 min), green liquor (6% total titratable alkali (TTA), 25% sulfidity based on TTA, 185 °C and 15 min), auto hydrolysis (185 °C, 30 min) and ozone delignification (25 °C, 30 min). Following the pretreatment, enzymatic saccharification was carried out using three enzyme charges of 10, 20 and 30 FPU per gram of substrate. The removal of lignin and hemicelluloses varied with the type of pretreatment and with the lignin content of the transgenic trees. High lignin content implied low enzymatic saccharification. Low xylan content native substrates lead to high enzymatic saccharification. High S to V (sryingaldehyde to vanillin) ratio substrates had high delignification during pretreatment. Compared to the wild type, the transgenics were better choice as feed stocks due to higher enzymatic saccharification without pretreatment which mean low the cost of bio-ethanol. Compared to three pretreatment methods, the green liquor pretreatment greatly improves the conversion of polysaccharides in general.}, number={8}, journal={BIOMASS & BIOENERGY}, author={Min, Douyong and Li, Quanzi and Jameel, Hasan and Chiang, Vincent and Chang, Hou-min}, year={2011}, month={Aug}, pages={3514–3521} }
 @article{lu_yang_chiang_2011, title={Conservation and Diversity of MicroRNA-associated Copper-regulatory Networks in Populus trichocarpa}, volume={53}, ISSN={["1744-7909"]}, DOI={10.1111/j.1744-7909.2011.01080.x}, abstractNote={Plants develop important regulatory networks to adapt to the frequently-changing availability of copper (Cu). However, little is known about miRNA-associated Cu-regulatory networks in plant species other than Arabidopsis. Here, we report that Cu-responsive miRNAs in Populus trichocarpa (Torr. & Gray) include not only conserved miR397, miR398 and miR408, but also Populus-specific miR1444, suggesting the conservation and diversity of Cu-responsive miRNAs in plants. Copper-associated suppression of mature miRNAs is in company with the up-regulation of their target genes encoding Cu-containing proteins in Populus. The targets include miR397-targeted PtLAC5, PtLAC6 and PtLAC110a, miR398-targeted PtCSD1, PtCSD2a and PtCSD2b, miR408-targeted PtPCL1, PtPCL2, PtPCL3 and PtLAC4, and miR1444-targeted PtPPO3 and PtPPO6. Consistently, P. trichocarpa miR408 promoter-directed GUS gene expression is down-regulated by Cu in transgenic tobacco plants. Cu-response elements (CuREs) are found in the promoters of Cu-responsive miRNA genes. We identified 34 SQUAMOSA-promoter binding protein-like (SPL) genes, of which 17 are full-length PtSPL proteins or partial sequences with at least 300 amino acids. Phylogenetic analysis indicates that PtSPL3 and PtSPL4 are CuRE-binding proteins controlling Cu-responsive gene expression. Cu appears to be not involved in the regulation of these transcription factors because neither PtSPL3 nor PtSPL4 is Cu-regulated and no CuRE exists in their promoters.}, number={11}, journal={JOURNAL OF INTEGRATIVE PLANT BIOLOGY}, author={Lu, Shanfa and Yang, Chenmin and Chiang, Vincent L.}, year={2011}, month={Nov}, pages={879–891} }
 @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{roque-rivera_talhelm_johnson_chiang_pregitzer_2011, title={Effects of lignin-modified Populus tremuloides on soil organic carbon}, volume={174}, ISSN={["1522-2624"]}, DOI={10.1002/jpln.201000445}, abstractNote={Abstract}, number={5}, journal={JOURNAL OF PLANT NUTRITION AND SOIL SCIENCE}, author={Roque-Rivera, Raysa and Talhelm, Alan F. and Johnson, Dale W. and Chiang, Vincent L. and Pregitzer, Kurt S.}, year={2011}, month={Oct}, pages={818–826} }
 @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} }
 @article{novaes_kirst_chiang_winter-sederoff_sederoff_2010, title={Lignin and Biomass: A Negative Correlation for Wood Formation and Lignin Content in Trees}, volume={154}, ISSN={["0032-0889"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77957730551&partnerID=MN8TOARS}, DOI={10.1104/pp.110.161281}, abstractNote={Studies in populations of forest tree hybrids have shown a negative correlation of biomass growth (usually measured as wood volume) and lignin content ([Kirst et al., 2004][1]; [Novaes et al., 2009][2]). The control of growth and lignin appears to be highly regulated, implying that selection for}, number={2}, journal={PLANT PHYSIOLOGY}, author={Novaes, Evandro and Kirst, Matias and Chiang, Vincent and Winter-Sederoff, Heike and Sederoff, Ronald}, year={2010}, month={Oct}, pages={555–561} }
 @article{suzuki_sakakibara_li_umezawa_chiang_2010, title={Profiling of phenylpropanoid monomers in developing xylem tissue of transgenic aspen (Populus tremuloides)}, volume={56}, ISSN={["1435-0211"]}, DOI={10.1007/s10086-009-1059-8}, abstractNote={Here we describe alterations in the cinnamate/monolignol pathway in three transgenic aspen lines: one with downregulated expression of 4-coumarate:CoA ligase (4CL), one with upregulated expression of coniferaldehyde 5-hydroxylase (CAld5H), and a 4CL downregulated/CAld5H upregulated line. Compared with the wild type, the 4CL downregulated line showed significantly increased levels of p-hydroxycinnamic acids such as p-coumaric, ferulic, and sinapic acids. In contrast, the CAld5H upregulated line had increased content of p-coumaryl and 5-hydroxyconiferyl alcohols. In the 4CL downregulated line, it was likely that most hydroxycinnamic acids were glycosylated. These results strongly suggest that the downregulation of 4CL and upregulation of CAld5H disrupt the metabolic flow through the cinnamate/monolignol pathway and thus alter the amount and structure of its final product, lignin.}, number={1}, journal={JOURNAL OF WOOD SCIENCE}, author={Suzuki, Shiro and Sakakibara, Norikazu and Li, Laigeng and Umezawa, Toshiaki and Chiang, Vincent L.}, year={2010}, month={Feb}, pages={71–76} }
 @article{shi_yang_lu_sederoff_chiang_2010, title={Specific down-regulation of PAL genes by artificial microRNAs in Populus trichocarpa}, volume={232}, ISSN={["0032-0935"]}, DOI={10.1007/s00425-010-1253-3}, abstractNote={Artificial microRNAs (amiRNAs) are similar to microRNAs (miRNAs) in that they are able to reduce the abundance of specific transcripts in plants by RNA-Induced Silencing Complex (RISC)-mediated cleavage and degradation, but differ in that they are designed for specific targets. The long generation times of forest trees have limited the discovery of mutations by conventional genetics. AmiRNAs can create gene-specific transcript reduction in transgenic trees in a single generation and may have broad application for functional genomics of trees. In this paper, we describe the specific down-regulation of multiple genes in the phenylalanine ammonia-lyase (PAL) gene family of Populus trichocarpa using amiRNA sequences incorporated in a P. trichocarpa miRNA-producing precursor, ptc-MIR408. Two different amiRNA constructs were designed to specifically down-regulate two different subsets of PAL genes, revealing differential regulation within the gene family. Down-regulation of subset A (PAL2, PAL4 and PAL5) by amiRNA-palA led to an increase in transcript abundance of subset B (PAL1 and PAL3). The reciprocal effect was not observed.}, number={6}, journal={PLANTA}, author={Shi, Rui and Yang, Chenmin and Lu, Shanfa and Sederoff, Ronald and Chiang, Vincent L.}, year={2010}, month={Nov}, pages={1281–1288} }
 @misc{shi_sun_li_heber_sederoff_chiang_2010, title={Towards a Systems Approach for Lignin Biosynthesis in Populus trichocarpa: Transcript Abundance and Specificity of the Monolignol Biosynthetic Genes}, volume={51}, ISSN={["1471-9053"]}, DOI={10.1093/pcp/pcp175}, abstractNote={As a step toward a comprehensive description of lignin biosynthesis in Populus trichocarpa, we identified from the genome sequence 95 phenylpropanoid gene models in 10 protein families encoding enzymes for monolignol biosynthesis. Transcript abundance was determined for all 95 genes in xylem, leaf, shoot and phloem using quantitative real-time PCR (qRT-PCR). We identified 23 genes that most probably encode monolignol biosynthesis enzymes during wood formation. Transcripts for 18 of the 23 are abundant and specific to differentiating xylem. We found evidence suggesting functional redundancy at the transcript level for phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), p-hydroxycinnamoyl-CoA:quinate shikimate p-hydroxycinnamoyltransferase (HCT), caffeoyl-CoA O-methyltransferase (CCoAOMT) and coniferyl aldehyde 5-hydroxylase (CAld5H). We carried out an enumeration-based motif identification and discriminant analysis on the promoters of all 95 genes. Five core motifs correctly discriminate the 18 xylem-specific genes from the 77 non-xylem genes. These motifs are similar to promoter elements known to regulate phenylpropanoid gene expression. This work suggests that genes in monolignol biosynthesis are regulated by multiple motifs, often related in sequence.}, number={1}, journal={PLANT AND CELL PHYSIOLOGY}, author={Shi, Rui and Sun, Ying-Hsuan and Li, Quanzi and Heber, Steffen and Sederoff, Ronald and Chiang, Vincent L.}, year={2010}, month={Jan}, pages={144–163} }
 @article{lu_sun_chiang_2009, title={Adenylation of plant miRNAs}, volume={37}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkp031}, abstractNote={The modification or degradation of RNAs including miRNAs may play vital roles in regulating RNA functions. The polyadenylation- and exosome-mediated RNA decay is involved in the degradation of plant RNAs including the primary miRNA processing intermediates. However, plant miRNA levels are not affected by exosome depletion. Here, we report the cloning of a large number of 5′ and/or 3′ truncated versions of the known miRNAs from various tissues of Populus trichocarpa (black cottonwood). It suggests that plant miRNAs may be degraded through either 5′ to 3′ or 3′ to 5′ exonucleolytic digestion. We also show that a significant portion of the isolated miRNAs contains, at the 3′-end, one or a few post-transcriptionally added adenylic acid residues, which are distinct in length from the polyadenylate tail added to other plant RNAs for exosome-mediated degradation. Using an in vitro miRNA degradation system, where synthesized miRNA oligos were degraded in extracts of P. trichocarpa cells, we revealed that the adenylated miRNAs were degraded slower than others without adenylation. It indicates that addition of adenylic acid residues on the 3′-end plays a negative role in miRNA degradation. Our results provide new information for understanding the mechanism of miRNA degradation.}, number={6}, journal={NUCLEIC ACIDS RESEARCH}, author={Lu, Shanfa and Sun, Ying-Hsuan and Chiang, Vincent L.}, year={2009}, month={Apr}, pages={1878–1885} }
 @misc{kumar_thammannagowda_bulone_chiang_han_joshi_mansfield_mellerowicz_sundberg_teeri_et al._2009, title={An update on the nomenclature for the cellulose synthase genes in Populus}, volume={14}, ISSN={["1878-4372"]}, DOI={10.1016/j.tplants.2009.02.004}, abstractNote={Cellulose synthase (CesA) is a central catalyst in the generation of the plant cell wall biomass and is, therefore, the focus of intense research. Characterization of individual CesA genes from Populus species has led to the publication of several different naming conventions for CesA gene family members in this model tree. To help reduce the resulting confusion, we propose here a new phylogeny-based CesA nomenclature that aligns the Populus CesA gene family with the established Arabidopsis thaliana CesA family structure.}, number={5}, journal={TRENDS IN PLANT SCIENCE}, author={Kumar, Manoj and Thammannagowda, Shivegowda and Bulone, Vincent and Chiang, Vincent and Han, Kyung-Hwan and Joshi, Chandrashekhar P. and Mansfield, Shawn D. and Mellerowicz, Ewa and Sundberg, Bjorn and Teeri, Tuula and et al.}, year={2009}, month={May}, pages={248–254} }
 @article{osakabe_osakabe_chiang_2009, title={Characterization of the tissue-specific expression of phenylalanine ammonia-lyase gene promoter from loblolly pine (Pinus taeda) in Nicotiana tabacum}, volume={28}, ISSN={["1432-203X"]}, DOI={10.1007/s00299-009-0707-1}, abstractNote={We isolated the 5' flanking region of a gene for phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Pinus taeda, PtaPAL. To investigate the tissue-specific expression of the PtaPAL promoter, histochemical assay of GUS activity was performed using the transgenic tobacco expressing the PtaPAL promoter-GUS. The region of -897 to -420 in PtaPAL promoter showed high activities in the secondary xylem and response to bending stress. To characterize the cis-regulatory functions of the promoters for enzymes in phenylpropanoid biosynthesis, we examined the activity of chimeric promoters of PtaPAL and a 4-coumarate CoA ligase, Pta4CL alpha. The chimeric promoter showed similar activity as the Pta4CL alpha promoter. Electrophoretic mobility shift assays implicated -897 to -674 of PtaPAL promoter containing cis-elements of the expression in xylem of Pinus taeda. The results suggested that AC elements of PtaPAL have multiple functions in the expression under the various developmental stages and stress conditions in the transgenic tobacco.}, number={9}, journal={PLANT CELL REPORTS}, author={Osakabe, Yuriko and Osakabe, Keishi and Chiang, Vincent L.}, year={2009}, month={Sep}, pages={1309–1317} }
 @article{osakabe_osakabe_chiang_2009, title={Isolation of 4-coumarate Co-A ligase gene promoter from loblolly pine (Pinus taeda) and characterization of tissue-specific activity in transgenic tobacco}, volume={47}, ISSN={["0981-9428"]}, DOI={10.1016/j.plaphy.2009.09.003}, abstractNote={We characterized promoter activity of a phenylpropanoid biosynthetic gene encoding 4-coumarate Co-A ligase (4CL), Pta4Clα, from Pinus taeda. Histochemical- and quantitative assays of GUS expression in the vascular tissue were performed using transgenic tobacco plants expressing promoter-GUS reporters. Deletion analysis of the Pta4Clα promoter showed that the region −524 to −252, which has two AC elements, controls the high expression levels in ray-parenchyma cells of older tobacco stems. High activity level of the promoter domain of Pta4CLα was also detected in the xylem cells under bending stress. DNA-protein complexes were detected in the reactions of the Pta4CLα promoter fragments with the nuclear proteins of xylem of P. taeda. The AC elements in the Pta4CLα promoter appeared to have individual roles during xylem development that are activated in a coordinated manner in response to stress in transgenic tobacco.}, number={11-12}, journal={PLANT PHYSIOLOGY AND BIOCHEMISTRY}, author={Osakabe, Yuriko and Osakabe, Keishi and Chiang, Vincent L.}, year={2009}, pages={1031–1036} }
 @article{schmidt_schwartzberg_perera_weber-bargioni_carroll_sarkar_bosneaga_urban_song_balakshin_et al._2009, title={Label-free in situ imaging of lignification in the cell wall of low lignin transgenic Populus trichocarpa}, volume={230}, ISSN={["1432-2048"]}, DOI={10.1007/s00425-009-0963-x}, abstractNote={Chemical imaging by confocal Raman microscopy has been used for the visualization of the cellulose and lignin distribution in wood cell walls. Lignin reduction in wood can be achieved by, for example, transgenic suppression of a monolignol biosynthesis gene encoding 4-coumarate-CoA ligase (4CL). Here, we use confocal Raman microscopy to compare lignification in wild type and lignin-reduced 4CL transgenic Populus trichocarpa stem wood with spatial resolution that is sub-μm. Analyzing the lignin Raman bands in the spectral region between 1,600 and 1,700 cm−1, differences in lignin signal intensity and localization are mapped in situ. Transgenic reduction of lignin is particularly pronounced in the S2 wall layer of fibers, suggesting that such transgenic approach may help overcome cell wall recalcitrance to wood saccharification. Spatial heterogeneity in the lignin composition, in particular with regard to ethylenic residues, is observed in both samples.}, number={3}, journal={PLANTA}, author={Schmidt, M. and Schwartzberg, A. M. and Perera, P. N. and Weber-Bargioni, A. and Carroll, A. and Sarkar, P. and Bosneaga, E. and Urban, J. J. and Song, J. and Balakshin, M. Y. and et al.}, year={2009}, month={Aug}, pages={589–597} }
 @article{sutela_niemi_edesi_laakso_saranpaa_vuosku_makela_tiimonen_chiang_koskimaki_et al._2009, title={Phenolic compounds in ectomycorrhizal interaction of lignin modified silver birch}, volume={9}, ISSN={["1471-2229"]}, DOI={10.1186/1471-2229-9-124}, abstractNote={The monolignol biosynthetic pathway interconnects with the biosynthesis of other secondary phenolic metabolites, such as cinnamic acid derivatives, flavonoids and condensed tannins. The objective of this study is to evaluate whether genetic modification of the monolignol pathway in silver birch (Betula pendula Roth.) would alter the metabolism of these phenolic compounds and how such alterations, if exist, would affect the ectomycorrhizal symbiosis.Silver birch lines expressing quaking aspen (Populus tremuloides L.) caffeate/5-hydroxyferulate O-methyltransferase (PtCOMT) under the 35S cauliflower mosaic virus (CaMV) promoter showed a reduction in the relative expression of a putative silver birch COMT (BpCOMT) gene and, consequently, a decrease in the lignin syringyl/guaiacyl composition ratio. Alterations were also detected in concentrations of certain phenolic compounds. All PtCOMT silver birch lines produced normal ectomycorrhizas with the ectomycorrhizal fungus Paxillus involutus (Batsch: Fr.), and the formation of symbiosis enhanced the growth of the transgenic plants.The down-regulation of BpCOMT in the 35S-PtCOMT lines caused a reduction in the syringyl/guaiacyl ratio of lignin, but no significant effect was seen in the composition or quantity of phenolic compounds that would have been caused by the expression of PtCOMT under the 35S or UbB1 promoter. Moreover, the detected alterations in the composition of lignin and secondary phenolic compounds had no effect on the interaction between silver birch and P. involutus.}, journal={BMC PLANT BIOLOGY}, author={Sutela, Suvi and Niemi, Karoliina and Edesi, Jaanika and Laakso, Tapio and Saranpaa, Pekka and Vuosku, Jaana and Makela, Riina and Tiimonen, Heidi and Chiang, Vincent L. and Koskimaki, Janne and et al.}, year={2009}, month={Sep} }
 @article{yeh_wang_rui_sun_chiang_2008, title={A novel O-Methyltransferase-like gene with a drastic extopic expression in response to tension wood formation in Poupulus trichocarpa}, volume={41}, number={9-10}, journal={Cellulose Chemistry and Technology}, author={Yeh, T. F. and Wang, J. and Rui, S. and Sun, Y. H. and Chiang, V. L.}, year={2008}, pages={521–528} }
 @article{lu_li_yi_joshi_chiang_2008, title={Differential expression of three eucalyptus secondary cell wall-related cellulose synthase genes in response to tension stress}, volume={59}, ISSN={["1460-2431"]}, DOI={10.1093/jxb/erm350}, abstractNote={Trees constitute the majority of lignocellulosic biomass existing on our planet. Trees also serve as important feedstock materials for various industrial products. However, little is known about the regulatory mechanisms of cellulose synthase (CesA) genes of trees. Here, the cloning and characterization of three CesA genes (EgraCesA1, EgraCesA2, and EgraCesA3) from an economically important tree species, Eucalyptus grandis, are reported. All three genes were specifically expressed in xylem cells of eucalyptus undergoing secondary cell wall biosynthesis. The GUS gene, expressed under the control of the EgraCesA2 or EgraCesA3 promoter, was also localized in the secondary xylem in transgenic tobacco stems. However, the EgraCesA1 promoter alone or along with its 5'-UTR introns was insufficient to direct appropriate GUS expression. EgraCesA2 and EgraCesA3 gene expression was up-regulated in tension-stressed eucalyptus xylem cells. Accordingly, GUS expression directed by the EgraCesA2 or EgraCesA3 promoter was also up-regulated. EgraCesA1 had no such response. Thus, it is most unlikely that EgraCesA1 is a subunit of the EgraCesA2-EgraCesA3 complex. The presence of at least two types of cellulose biosynthesis machinery in wood formation is an important clue in deciphering the underpinnings of the perennial growth of trees in various environmental conditions. By analysing GUS gene expression directed by the EgraCesA3 promoter or its deletions, several negative and positive regulatory regions controlling gene expression in xylem or phloem were identified. Also a region which is likely to contain mechanical stress-responsive elements was deduced. These results will guide further studies on identifying cis-regulatory elements directing CesA gene transcription and wood formation regulatory networks.}, number={3}, journal={JOURNAL OF EXPERIMENTAL BOTANY}, author={Lu, Shanfa and Li, Laigeng and Yi, Xiaoping and Joshi, Chandrashekhar P. and Chiang, Vincent L.}, year={2008}, month={Feb}, pages={681–695} }
 @article{tiimonen_aronen_laakso_saranpaa_chiang_haggman_niemi_2008, title={Paxillus involutus Forms an Ectomycorrhizal Symbiosis and Enhances Survival of PtCOMT-modified Betula pendula in vitro}, volume={57}, ISSN={["2509-8934"]}, DOI={10.1515/sg-2008-0036}, abstractNote={Abstract}, number={4-5}, journal={SILVAE GENETICA}, author={Tiimonen, H. and Aronen, T. and Laakso, T. and Saranpaa, P. and Chiang, V. and Haggman, H. and Niemi, K.}, year={2008}, pages={235–242} }
 @article{nakatsubo_li_hattori_lu_sakakibara_chiang_shimada_suzuki_umezawa_2008, title={Roles of 5-hydroxyconiferylaldehyde and caffeoyl CoA O-methyltransferases in monolignol biosynthesis in Carthamus tinctorius}, volume={41}, number={9-10}, journal={Cellulose Chemistry and Technology}, author={Nakatsubo, T. and Li, L. and Hattori, T. and Lu, S. and Sakakibara, N. and Chiang, V. L. and Shimada, M. and Suzuki, S. and Umezawa, T.}, year={2008}, pages={511–520} }
 @article{lu_sun_chiang_2008, title={Stress-responsive microRNAs in Populus}, volume={55}, ISSN={["1365-313X"]}, DOI={10.1111/j.1365-313X.2008.03497.x}, abstractNote={Summary}, number={1}, journal={PLANT JOURNAL}, author={Lu, Shanfa and Sun, Ying-Hsuan and Chiang, Vincent L.}, year={2008}, month={Jul}, pages={131–151} }
 @misc{lu_sun_amerson_chiang_2007, title={MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development}, volume={51}, ISSN={["1365-313X"]}, DOI={10.1111/j.1365-313x.2007.03208.x}, abstractNote={Summary}, number={6}, journal={PLANT JOURNAL}, author={Lu, Shanfa and Sun, Ying-Hsuan and Amerson, Henry and Chiang, Vincent L.}, year={2007}, month={Sep}, pages={1077–1098} }
 @inproceedings{keshwani_cheng_li_burns_chiang_2007, title={Microwave pretreatment of switchgrass to enhance enzymatic hydrolysis}, volume={077127}, DOI={10.13031/2013.23472}, abstractNote={Switchgrass is a promising lignocellulosic biomass for fuel-ethanol production. However, pretreatment of lignocellulosic materials is necessary to improve its susceptibility to enzymatic hydrolysis. The objectives of this study were to examine the feasibility of microwave pretreatment to enhance enzymatic hydrolysis of switchgrass and to determine the optimal pretreatment conditions. Switchgrass samples immersed in water, dilute sulfuric acid and dilute sodium hydroxide solutions were exposed to microwave radiation at varying levels of radiation power and residence time. Pretreated solids were enzymatically hydrolyzed and reducing sugars in the hydrolysate were analyzed. Microwave radiation of switchgrass at lower power levels resulted in more efficient enzymatic hydrolysis. The application of microwave radiation for 10 minutes at 250 watts to switchgrass immersed in 3% sodium hydroxide solution (w/v) produced the highest yields of reducing sugar. Results were comparable to conventional 60 minute sodium hydroxide pretreatment of switchgrass. The findings suggest that combined microwave-alkali is a promising pretreatment method to enhance enzymatic hydrolysis of switchgrass.}, booktitle={Proceedings of the ASABE Annual International Meeting (Minneapolis, Minnesota)}, author={Keshwani, D. R. and Cheng, J. J. and Li, L. and Burns, J. C. and Chiang, V.}, year={2007} }
 @article{hancock_loya_giardina_li_chiang_pregitzer_2007, title={Plant growth, biomass partitioning and soil carbon formation in response to altered lignin biosynthesis in Populus tremuloides}, volume={173}, ISSN={["1469-8137"]}, DOI={10.1111/j.1469-8137.2006.01965.x}, abstractNote={We conducted a glasshouse mesocosm study that combined (13)C isotope techniques with wild-type and transgenic aspen (Populus tremuloides) in order to examine how altered lignin biosynthesis affects plant production and soil carbon formation. Our transgenic aspen lines expressed low stem lignin concentration but normal cellulose concentration, low lignin stem concentration with high cellulose concentration or an increased stem syringyl to guaiacyl lignin ratio. Large differences in stem lignin concentration observed across lines were not observed in leaves or fine roots. Nonetheless, low lignin lines accumulated 15-17% less root C and 33-43% less new soil C than the control line. Compared with the control line, transformed aspen expressing high syringyl lignin accumulated 30% less total plant C - a result of greatly reduced total leaf area - and 70% less new soil C. These findings suggest that altered stem lignin biosynthesis in Populus may have little effect on the chemistry of fine roots or leaves, but can still have large effects on plant growth, biomass partitioning and soil C formation.}, number={4}, journal={NEW PHYTOLOGIST}, author={Hancock, Jessica E. and Loya, Wendy M. and Giardina, Christian P. and Li, Laigeng and Chiang, Vincent L. and Pregitzer, Kurt S.}, year={2007}, pages={732–742} }
 @article{tiimonen_haggman_tsai_chiang_aronen_2007, title={The seasonal activity and the effect of mechanical bending and wounding on the PtCOMT promoter in Betula pendula Roth}, volume={26}, ISSN={["1432-203X"]}, DOI={10.1007/s00299-007-0331-x}, abstractNote={In this study, 900-bp (signed as p including nucleotides -1 to -886) and partly deleted (signed as dp including nucleotides -1 to -414) COMT (caffeate/5-hydroxyferulate O-methyltransferase) promoters from Populus tremuloides Michx. were fused to the GUS reporter gene, and the tissue-specific expression patterns of the promoters were determined in Betula pendula Roth along the growing season, and as a response to mechanical bending and wounding. The main activity of the PtCOMTp- and PtCOMTdp-promoters, determined by the histochemical GUS assay, was found in the developing xylem of stems during the 8th-13th week and in the developing xylem of roots in the 13th week of the growing season. The GUS expression patterns did not differ among the xylem cell types. The PtCOMT promoter-induced GUS expression observed in phloem fibres suggests a need for PtCOMT expression and thus syringyl (S) lignin synthesis in fibre lignification. However, the PtCOMTdp-promoter induced GUS expression in stem trichomes, which may contribute to the biosynthesis of phenylpropanoid pathway-derived compounds other than lignin. Finally, a strong GUS expression was induced by the PtCOMT promoters in response to mechanical stem bending but not to wounding. The lack of major differences between the PtCOMTp- and PtCOMTdp-promoters suggests that the deleted promoter sequence (including nucleotides -415 to -886) did not contain a significant regulatory element contributing to the GUS expression in young B. pendula trees.}, number={8}, journal={PLANT CELL REPORTS}, author={Tiimonen, Heidi and Haggman, Hely and Tsai, Chung-Jui and Chiang, Vincent and Aronen, Tuija}, year={2007}, month={Aug}, pages={1205–1214} }
 @misc{li_lu_chiang_2006, title={A genomic and molecular view of wood formation}, volume={25}, ISSN={["1549-7836"]}, DOI={10.1080/07352680600611519}, abstractNote={Wood formation is a process derived from plant secondary growth. Different from primary growth, plant secondary growth is derived from cambium meristem cells in the vascular and cork cambia and leads to the girth increase of the plant trunk. In the secondary growth process, plants convert most of photosynthesized products into various biopolymers for use in the formation of woody tissues. This article summarizes the new developments of genomic and genetic characterization of wood formation in herbaceous model plant and tree plant systems. Genomic studies have categorized a collection of the genes for which expression is associated with secondary growth. During wood formation, the expression of many genes is regulated in a stage-specific manner. The function of many genes involved in wood biosyntheses and xylem differentiation has been characterized. Although great progress has been achieved in the molecular and genomic understanding of plant secondary growth in recent years, the profound genetic mechanisms underlying this plant development remain to be investigated. Completion of the first tree genome sequence (Populus genome) provides a valuable genomic resource for characterization of plant secondary growth.}, number={3}, journal={CRITICAL REVIEWS IN PLANT SCIENCES}, author={Li, Laigeng and Lu, Shanfa and Chiang, Vincent}, year={2006}, pages={215–233} }
 @article{lu_zhou_li_chiang_2006, title={Distinct roles of cinnamate 4-hydroxylase genes in Populus}, volume={47}, ISSN={["1471-9053"]}, DOI={10.1093/pcp/pcj063}, abstractNote={Cinnamate 4-hydroxylase (C4H) catalyzes the conversion of cinnamate into 4-hydroxy-cinnamate, a key reaction of the phenylpropanoid pathway which leads to the biosynthesis of several secondary metabolites. C4H genes exist as a multigene family in various plant species. In order to understand the roles of individual C4H members, four C4H cDNAs (PtreC4H) were isolated from Populus tremuloides and three C4H loci (PtriC4H) were identified in the P. trichocarpa genome. The ability of Populus C4H isoforms to convert trans-cinnamate into p-coumaric acid was verified by the examination of yeast recombinant PtreC4H proteins. Populus C4H genes were expressed in various tissues, including developing xylem, phloem and epidermis; however, the expression patterns of individual members were different from each other. Sequential analysis of C4H promoters showed that the differential expression of C4H genes was associated with cis-acting regulatory elements such as box L, box P and H box, suggesting that the divergent C4H isoforms played distinct roles in the production of secondary metabolites. The involvement of specific C4H isoforms in the biosynthesis of guaiacyl and syringyl monolignols is discussed.}, number={7}, journal={PLANT AND CELL PHYSIOLOGY}, author={Lu, Shanfa and Zhou, Yihua and Li, Laigeng and Chiang, Vincent L.}, year={2006}, month={Jul}, pages={905–914} }
 @article{song_lu_chen_lourenco_chiang_2006, title={Genetic transformation of Populus trichocarpa genotype Nisqually-1: A functional genomic tool for woody plants}, volume={47}, ISSN={["1471-9053"]}, DOI={10.1093/pcp/pcl018}, abstractNote={We report here the Agrobacterium-mediated genetic transformation of Nisqually-1, a Populus trichocarpa genotype whose genome was recently sequenced. Several systems were established. Internodal stem segments from vigorously growing greenhouse plants are the explants most amenable to transformation. For the most efficient system, approximately 40% of the stem segments infected with pBI121-containing Agrobacterium tumefaciens C58 produced transgenic calli, as confirmed by beta-glucuronidase (GUS) staining. The regeneration efficiency of independent transgenic plants was approximately 13%, as revealed by genomic Southern analysis. Some transgenic plants were produced in as little as 5 months after co-cultivation. This system may help to facilitate studies of gene functions in tree growth and development at a genome level.}, number={11}, journal={PLANT AND CELL PHYSIOLOGY}, author={Song, Jingyuan and Lu, Shanfa and Chen, Zenn-Zong and Lourenco, Rodrigo and Chiang, Vincent L.}, year={2006}, month={Nov}, pages={1582–1589} }
 @article{chiang_2006, title={Monolignol biosynthesis and genetic engineering of lignin in trees, a review}, volume={4}, ISSN={["1610-3653"]}, DOI={10.1007/s10311-006-0067-9}, number={3}, journal={ENVIRONMENTAL CHEMISTRY LETTERS}, author={Chiang, Vincent L.}, year={2006}, month={Aug}, pages={143–146} }
 @article{yamada_yeh_chang_li_kadla_chiang_2006, title={Rapid analysis of transgenic trees using transmittance near-infrared spectroscopy (NIR)}, volume={60}, ISSN={["1437-434X"]}, DOI={10.1515/HF.2006.005}, abstractNote={Abstract}, number={1}, journal={HOLZFORSCHUNG}, author={Yamada, T and Yeh, TF and Chang, HM and Li, LG and Kadla, JF and Chiang, VL}, year={2006}, month={Jan}, pages={24–28} }
 @article{suzuki_li_sun_chiang_2006, title={The cellulose synthase gene superfamily and biochemical functions of xylem-specific cellulose synthase-like genes in Populus trichocarpa}, volume={142}, ISSN={["1532-2548"]}, DOI={10.1104/pp.106.086678}, abstractNote={Abstract}, number={3}, journal={PLANT PHYSIOLOGY}, author={Suzuki, Shiro and Li, Laigeng and Sun, Ying-Hsuan and Chiang, Vincent L.}, year={2006}, month={Nov}, pages={1233–1245} }
 @article{li_cheng_lu_nakatsubo_umezawa_chiang_2005, title={Clarification of cinnamoyl co-enzyme a reductase catalysis in monolignol biosynthesis of aspen}, volume={46}, ISSN={["1471-9053"]}, DOI={10.1093/pcp/pci120}, abstractNote={Cinnamoyl co-enzyme A reductase (CCR), one of the key enzymes involved in the biosynthesis of monolignols, has been thought to catalyze the conversion of several cinnamoyl-CoA esters to their respective cinnamaldehydes. However, it is unclear which cinnamoyl-CoA ester is metabolized for monolignol biosynthesis. A xylem-specific CCR cDNA was cloned from aspen (Populus tremuloides) developing xylem tissue. The recombinant CCR protein was produced through an Escherichia coli expression system and purified to electrophoretic homogeneity. The biochemical properties of CCR were characterized through direct structural corroboration and quantitative analysis of the reaction products using a liquid chromatography-mass spectrometry system. The enzyme kinetics demonstrated that CCR selectively catalyzed the reduction of feruloyl-CoA from a mixture of five cinnamoyl CoA esters. Furthermore, feruloyl-CoA showed a strong competitive inhibition of the CCR catalysis of other cinnamoyl CoA esters. Importantly, when CCR was coupled with caffeoyl-CoA O-methyltransferase (CCoAOMT) to catalyze the substrate caffeoyl-CoA ester, coniferaldehyde was formed, suggesting that CCoAOMT and CCR are neighboring enzymes. However, the in vitro results also revealed that the reactions mediated by these two neighboring enzymes require different pH environments, indicating that compartmentalization is probably needed for CCR and CCoAOMT to function properly in vivo. Eight CCR homologous genes were identified in the P. trichocarpa genome and their expression profiling suggests that they may function differentially.}, number={7}, journal={PLANT AND CELL PHYSIOLOGY}, author={Li, LG and Cheng, XF and Lu, SF and Nakatsubo, T and Umezawa, T and Chiang, VL}, year={2005}, month={Jul}, pages={1073–1082} }
 @article{tiimonen_aronen_laakso_saranpaa_chiang_ylioja_roininen_haggman_2005, title={Does lignin modification affect feeding preference or growth performance of insect herbivores in transgenic silver birch (Betula pendula Roth)?}, volume={222}, ISSN={["1432-2048"]}, DOI={10.1007/s00425-005-0002-5}, abstractNote={Transgenic silver birch (Betula pendula Roth) lines were produced in order to modify lignin biosynthesis. These lines carry COMT (caffeate/5-hydroxyferulate O-methyltransferase) gene from Populus tremuloides driven by constitutive promoter 35S CaMV (cauliflower mosaic virus) or UbB1 (ubiquitin promoter from sunflower). The decreased syringyl/guaiacyl (S/G) ratio was found in stem and leaf lignin of 35S CaMV-PtCOMT transgenic silver birch lines when compared to non-transformed control or UbB1-PtCOMT lines. In controlled feeding experiments the leaves of transgenic birch lines as well as controls were fed to insect herbivores common in boreal environment, i.e., larvae of Aethalura punctulata, Cleora cinctaria and Trichopteryx carpinata (Lepidoptera: Geometridae) as well as the adults of birch leaf-feeding beetles Agelastica alni (Coleoptera: Chrysomelidae) and Phyllobius spp. (Coleoptera: Curculionidae). The feeding preferences of these herbivores differed in some cases among the tested birch lines, but these differences could not be directly associated to lignin modification. They could as well be explained by other characteristics of leaves, either natural or caused by transgene site effects. Growth performance of lepidopteran larvae fed on transgenic or control leaves did not differ significantly.}, number={4}, journal={PLANTA}, author={Tiimonen, H and Aronen, T and Laakso, T and Saranpaa, P and Chiang, V and Ylioja, T and Roininen, H and Haggman, H}, year={2005}, month={Nov}, pages={699–708} }
 @article{shi_chiang_2005, title={Facile means for quantifying microRNA expression by real-time PCR}, volume={39}, ISSN={["1940-9818"]}, DOI={10.2144/000112010}, abstractNote={ MicroRNAs (miRNAs) are 20–24 nucleotide RNAs that are predicted to play regulatory roles in animals and plants. Here we report a simple and sensitive real-time PCR method for quantifying the expression of plant miRNAs. Total RNA, including miRNAs, was polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer. Several Arabidopsis miRNA sequences were tested using SYBR® Green reagent, demonstrating that this method, using as little as 100 pg total RNA, could readily discriminate the expression of miRNAs having as few as one nucleotide sequence difference. This method also revealed miRNA tissue-specific expression patterns that cannot be resolved by Northern blot analysis and may therefore be widely useful for characterizing miRNA expression in plants as well as in animals. }, number={4}, journal={BIOTECHNIQUES}, author={Shi, R and Chiang, VL}, year={2005}, month={Oct}, pages={519–525} }
 @misc{chiang_li_2005, title={Methods for simultaneous control of lignin content and composition, and cellulose content in plants}, volume={6855864}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Chiang, V. L. C. and Li, L.}, year={2005} }
 @article{lu_sun_shi_clark_li_chiang_2005, title={Novel and mechanical stress-responsive microRNAs in Populus trichocarpa that are absent from Arabidopsis}, volume={17}, DOI={10.1105/tpc.105.033456}, abstractNote={Abstract}, number={8}, journal={Plant Cell}, author={Lu, S. F. and Sun, Y. H. and Shi, R. and Clark, C. and Li, L. G. and Chiang, V. L.}, year={2005}, pages={2186–2203} }
 @article{yeh_yamada_capanema_chang_chiang_kadla_2005, title={Rapid screening of wood chemical component variations using transmittance near-infrared spectroscopy}, volume={53}, ISSN={["1520-5118"]}, DOI={10.1021/jf0480647}, abstractNote={A rapid transmittance near-infrared (NIR) spectroscopy method was developed to predict the variation in chemical composition of solid wood. The effect of sample preparation, sample quantity (single versus stacked multiple wood wafers), and NIR acquisition time on the quantification of alpha-cellulose and lignin content was investigated. Strong correlations were obtained between laboratory wet chemistry values and the NIR-predicted values. In addition to the experimental protocol and method development, improvements in calibration error associated with utilizing stacked multiple wood wafers as opposed to single wood wafers are also discussed.}, number={9}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Yeh, TF and Yamada, T and Capanema, E and Chang, HM and Chiang, V and Kadla, JF}, year={2005}, month={May}, pages={3328–3332} }
 @misc{chiang_li_2004, title={Genetic engineering of syringyl-enriched lignin in plants}, volume={6812377}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Chiang, V. L. C. and Li, L.}, year={2004} }
 @article{lu_shi_tsao_yi_li_chiang_2004, title={RNA silencing in plants by the expression of siRNA duplexes}, volume={32}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gnh170}, abstractNote={In animal cells, stable RNA silencing can be achieved by vector-based small interfering RNA (siRNA) expression system, in which Pol III RNA gene promoters are used to drive the expression of short hairpin RNA, however, this has not been demonstrated in plants. Whether Pol III RNA gene promoter is capable of driving siRNA expression in plants is unknown. Here, we report that RNA silencing was achieved in plants through stable expression of short hairpin RNA, which was driven by Pol III RNA gene promoters. Using glucuronidase (GUS) transformed tobacco as a model system, the results demonstrated that 21 nt RNA duplexes, targeting at different sites of GUS gene, were stably expressed under the control of either human H1 or Arabidopsis 7SL RNA gene promoter, and GUS gene was silenced in 80% of siRNA transgenics. The severity of silencing was correlated with the abundance of siRNA expression but independent of the target sites and uridine residue structures in siRNA hairpin transcripts. Thus, the specific expression of siRNA provides a new system for the study of siRNA silencing pathways and functional genomics in plants. Moreover, the effectiveness of the human H1 promoter in a plant background suggested a conserved mechanism underlying Pol III complex functionality.}, number={21}, journal={NUCLEIC ACIDS RESEARCH}, author={Lu, SF and Shi, R and Tsao, CC and Yi, XP and Li, LG and Chiang, VL}, year={2004} }
 @article{li_zhou_cheng_sun_marita_ralph_chiang_2003, title={Combinatorial modification of multiple lignin traits in trees through multigene cotransformation}, volume={100}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0831166100}, abstractNote={
            Lignin quantity and reactivity [which is associated with its syringyl/guaiacyl (S/G) constituent ratio] are two major barriers to wood-pulp production. To verify our contention that these traits are regulated by distinct monolignol biosynthesis genes, encoding 4-coumarate–CoA ligase (4CL) and coniferaldehyde 5-hydroxylase (CAld5H), we used
            Agrobacterium
            to cotransfer antisense
            4CL
            and sense
            CAld5H
            genes into aspen (
            Populus tremuloides
            ). Trees expressing each one and both of the transgenes were produced with high efficiency. Lignin reduction by as much as 40% with 14% cellulose augmentation was achieved in antisense
            4CL
            plants; S/G-ratio increases as much as 3-fold were observed without lignin quantity change in sense
            CAld5H
            plants. Consistent with our contention, these effects were independent but additive, with plants expressing both transgenes having up to 52% less lignin, a 64% higher S/G ratio, and 30% more cellulose. An S/G-ratio increase also accelerated cell maturation in stem secondary xylem, pointing to a role for syringyl lignin moieties in coordinating xylem secondary wall biosynthesis. The results suggest that this multigene cotransfer system should be broadly useful for plant genetic engineering and functional genomics.
          }, number={8}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Li, L and Zhou, YH and Cheng, XF and Sun, JY and Marita, JM and Ralph, J and Chiang, VL}, year={2003}, month={Apr}, pages={4939–4944} }
 @article{harding_leshkevich_chiang_tsai_2002, title={Differential substrate inhibition couples kinetically distinct 4-coumarate: Coenzyme A ligases with spatially distinct metabolic roles in quaking aspen}, volume={128}, DOI={10.1104/pp.010603}, abstractNote={Abstract}, number={2002}, journal={Plant Physiology}, author={Harding, S. A. and Leshkevich, J. and Chiang, V. L. and Tsai, C. J.}, year={2002}, pages={428–438} }
 @article{chiang_2002, title={From rags to riches}, volume={20}, ISSN={["1087-0156"]}, DOI={10.1038/nbt0602-557}, number={6}, journal={NATURE BIOTECHNOLOGY}, author={Chiang, VL}, year={2002}, month={Jun}, pages={557–558} }
 @misc{chiang_tsai_hu_2002, title={Methods of modifying lignin in plants by transformation with a 4-coumarate coenzyme a ligase nucleic acid}, volume={6455762}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Chiang, V. L. and Tsai, C.-J. and Hu, W.-J.}, year={2002} }
 @misc{chiang_tsai_podila_1999, title={Genetic engineering of wood color in plants}, volume={5886243}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Chiang, V. L. and Tsai, C. J. and Podila, G. K.}, year={1999} }