A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.

We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa secondary differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. A total of 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the secondary differentiating xylem. This extensive experimental data set, generated from a single tissue specialized in wood formation, was used to construct the predictive kinetic metabolic-flux model to provide a comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a longstanding paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides an explanation of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants.

ORCID IDs: 0000-0002-5392-0076 (J.P.W.); 0000-0001-7120-4690 (Y.-C.L.); 0000-0002-3021-3942 (J.D.); 0000-0002-7152-9601 (V.L.C.)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.

4-Coumaric acid:coenzyme A ligase (4CL) is involved in monolignol biosynthesis for lignification in plant cell walls. It ligates coenzyme A (CoA) with hydroxycinnamic acids, such as 4-coumaric and caffeic acids, into hydroxycinnamoyl-CoA thioesters. The ligation ensures the activated state of the acid for reduction into monolignols. In Populus spp., it has long been thought that one monolignol-specific 4CL is involved. Here, we present evidence of two monolignol 4CLs, Ptr4CL3 and Ptr4CL5, in Populus trichocarpa. Ptr4CL3 is the ortholog of the monolignol 4CL reported for many other species. Ptr4CL5 is novel. The two Ptr4CLs exhibited distinct Michaelis-Menten kinetic properties. Inhibition kinetics demonstrated that hydroxycinnamic acid substrates are also inhibitors of 4CL and suggested that Ptr4CL5 is an allosteric enzyme. Experimentally validated flux simulation, incorporating reaction/inhibition kinetics, suggested two CoA ligation paths in vivo: one through 4-coumaric acid and the other through caffeic acid. We previously showed that a membrane protein complex mediated the 3-hydroxylation of 4-coumaric acid to caffeic acid. The demonstration here of two ligation paths requiring these acids supports this 3-hydroxylation function. Ptr4CL3 regulates both CoA ligation paths with similar efficiencies, whereas Ptr4CL5 regulates primarily the caffeic acid path. Both paths can be inhibited by caffeic acid. The Ptr4CL5-catalyzed caffeic acid metabolism, therefore, may also act to mitigate the inhibition by caffeic acid to maintain a proper ligation flux. A high level of caffeic acid was detected in stemdifferentiating xylem of P. trichocarpa. Our results suggest that Ptr4CL5 and caffeic acid coordinately modulate the CoA ligation flux for monolignol biosynthesis.Lignin is an irreversible, terminal output of a major metabolic pathway in plant secondary metabolism. It is a polyphenolic structural component of the secondary cell walls of vascular plants and plays a unique role in the adaptation of plants to their environment and in resistance to biotic and abiotic stresses (Harada and Cote, 1985). In the formation of secondary cell walls, lignin is deposited between cells (middle lamella) and in cell walls. In the secondary wall, lignin surrounds the hemicelluloses and cellulose, forming a composite of the three major wall components (Timell, 1969;Terashima et al., 2009). Lignin is a major barrier to the utilization of biomass for energy, for papermaking, and for forage digestibility due to its interaction with cellulosics (Sarkanen, 1976;Chiang, 2002;Chen and Dixon, 2007). Studying lignin biosynthesis at the systems or holistic level should lead to better understanding of how the entire biosynthetic pathway is organized and regulated, providing more precise strategies to improve the production of energy, biomaterials, and food.Lignin is typically polymerized from three phenylpropanoid monomers, 4-coumaryl alcohol (metabolite 20 in Fig. 1), coniferyl alcohol (22), and sinapyl alcohol (24), also called the H, G, and S mo...

Identifying the transcription factors (TFs) and associated networks involved in stem cell regulation is essential for understanding the initiation and growth of plant tissues and organs. Although many TFs have been shown to have a role in the Arabidopsis root stem cells, a comprehensive view of the transcriptional signature of the stem cells is lacking. In this work, we used spatial and temporal transcriptomic data to predict interactions among the genes involved in stem cell regulation. To accomplish this, we transcriptionally profiled several stem cell populations and developed a gene regulatory network inference algorithm that combines clustering with dynamic Bayesian network inference. We leveraged the topology of our networks to infer potential major regulators. Specifically, through mathematical modeling and experimental validation, we identified PERIANTHIA (PAN) as an important molecular regulator of quiescent center function. The results presented in this work show that our combination of molecular biology, computational biology, and mathematical modeling is an efficient approach to identify candidate factors that function in the stem cells.root stem cell | root development | cell-type expression profile | gene regulatory network | modeling I dentifying the transcriptional signature underlying stem cell regulation is fundamental to understanding the initiation and growth of plant tissues and organs. The Arabidopsis thaliana root provides a tractable system to study stem cells since they are spatially confined at the tip of the root, in the stem cell niche (SCN), and are anatomically well characterized. The SCN contains several stem cell populations that include the cortexendodermis initials (CEIs), vascular initials [including phloem and xylem (XYL)], columella initials, and epidermal/lateral root cap initials. These stem cell populations divide asymmetrically to replenish the stem cell and produce a daughter cell that later differentiates into the different tissues of the root. In the center of all of these stem cell populations is the quiescent center (QC), which acts as the organizing center and maintains the surrounding stem cells in an undifferentiated state (1). Major players in stemcell regulation have been previously identified, such as BABY BOOM (BBM) and PLETHORA1-3 (PLT1, PLT2, PLT3/AIL6), which are important for proper root formation and maintenance (2). Additionally in the QC, the homeodomain transcription factor WUSCHEL-RELATED HOMEOBOX 5 acts noncell autonomously to maintain the columella stem cells (3-5). In the endodermal and cortical layers, the GRAS family transcription factors SHORTROOT (SHR) and SCARECROW (SCR) activate the transcription of the cell-cycle gene CYCLIN D6 (CYCD6) to trigger the asymmetric cell division of the CEI (6, 7). Additional TFs, such as REVOLUTA (REV) and PHABULOSA (PHB), have been shown to regulate tissue specification and differentiation in the vascular stem cells (8-11). Despite these findings, a transcriptional signature within and across each of these diff...