Ectopic Expression of PtrLBD39 Retarded Primary and Secondary Growth in Populus trichocarpa.

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.

Woody plant cell walls: Fundamentals and utilization.

Cell walls in plants, particularly forest trees, are the major carbon sink of the terrestrial ecosystem. Chemical and biosynthetic features of plant cell walls were revealed early on, focusing mostly on herbaceous model species. Recent developments in genomics, transcriptomics, epigenomics, transgenesis, and associated analytical techniques are enabling novel insights into formation of woody cell walls. Here, we review multilevel regulation of cell wall biosynthesis in forest tree species. We highlight current approaches to engineering cell walls as potential feedstock for materials and energy and survey reported field tests of such engineered transgenic trees. We outline opportunities and challenges in future research to better understand cell type biogenesis for more efficient wood cell wall modification and utilization for biomaterials or for enhanced carbon capture and storage.

Multiplex CRISPR editing of wood for sustainable fiber production.

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.

PtrVCS2 Regulates Drought Resistance by Changing Vessel Morphology and Stomatal Closure in Populus trichocarpa.

Drought has severe effects on plant growth, forest productivity, and survival throughout the world. Understanding the molecular regulation of drought resistance in forest trees can enable effective strategic engineering of novel drought-resistant genotypes of tree species. In this study, we identified a gene, PtrVCS2, encoding a zinc finger (ZF) protein of the ZF-homeodomain transcription factor in Populus trichocarpa (Black Cottonwood) Torr. & A. Gray. ex Hook. Overexpression of PtrVCS2 (OE-PtrVCS2) in P. trichocarpa resulted in reduced growth, a higher proportion of smaller stem vessels, and strong drought-resistance phenotypes. Stomatal movement experiments revealed that the OE-PtrVCS2 transgenics showed lower stomata apertures than wild-type plants under drought conditions. RNA-seq analysis of the OE-PtrVCS2 transgenics showed that PtrVCS2 regulates the expression of multiple genes involved in regulation of stomatal opening and closing, particularly the PtrSULTR3;1-1 gene, and several genes related to cell wall biosynthesis, such as PtrFLA11-12 and PtrPR3-3. Moreover, we found that the water use efficiency of the OE-PtrVCS2 transgenic plants was consistently higher than that of wild type plants when subjected to chronic drought stress. Taken together, our results suggest that PtrVCS2 plays a positive role in improving drought adaptability and resistance in P. trichocarpa.

Cell-type-specific PtrWOX4a and PtrVCS2 form a regulatory nexus with a histone modification system for stem cambium development in Populus trichocarpa.

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.

The Manchurian Walnut Genome: Insights into Juglone and Lipid Biosynthesis.

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.

Dimerization of PtrMYB074 and PtrWRKY19 mediates transcriptional activation of PtrbHLH186 for secondary xylem development in Populus trichocarpa.

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.

A PtrLBD39-mediated transcriptional network regulates tension wood formation in Populus trichocarpa.

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.

Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa.

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.

Molecular and Metabolic Insights into Anthocyanin Biosynthesis for Leaf Color Change in Chokecherry (Padus virginiana).

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.

Histone Acetylation Changes in Plant Response to Drought Stress.

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.

CRISPR-Cas9 editing of CAFFEOYL SHIKIMATE ESTERASE 1 and 2 shows their importance and partial redundancy in lignification in Populus tremula × P. alba.

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.

MYB-Mediated Regulation of Anthocyanin Biosynthesis.

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.

Transcriptional reprogramming of xylem cell wall biosynthesis in tension wood.

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.

The microRNA476a-RFL module regulates adventitious root formation through a mitochondria-dependent pathway in Populus.

For woody plants, clonal propagation efficiency is largely determined by adventitious root (AR) formation at the bases of stem cuttings. However, our understanding of the molecular mechanisms contributing to AR morphogenesis in trees remains limited, despite the importance of vegetative propagation, currently the most common practice for tree breeding and commercialization. Here, we identified Populus-specific miR476a as a regulator of wound-induced adventitious rooting that acts by orchestrating mitochondrial homeostasis. MiR476a exhibited inducible expression during AR formation and directly targeted several Restorer of Fertility like (RFL) genes encoding mitochondrion-localized pentatricopeptide repeat proteins. Genetic modification of miR476a-RFL expression revealed that miR476a/RFL-mediated dynamic regulation of mitochondrial homeostasis influences AR formation in poplar. Mitochondrial perturbation via exogenous application of a chemical inhibitor indicated that miR476a/RFL-directed AR formation depends on mitochondrial regulation that acts via auxin signaling. Our results thus establish a microRNA-directed mitochondrion-auxin signaling cascade required for AR development, providing insights into the role of mitochondrial regulation in the developmental plasticity of plants.

A multiscale model of lignin biosynthesis for predicting bioenergy traits in Populus trichocarpa.

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.

Microbial Interactions Within Multiple-Strain Biological Control Agents Impact Soil-Borne Plant Disease.

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.

MYB Transcription Factor161 Mediates Feedback Regulation of Secondary wall-associated NAC-Domain1 Family Genes for Wood Formation.

Wood formation is a complex process that involves cell differentiation, cell expansion, secondary wall deposition, and programmed cell death. We constructed a four-layer wood formation transcriptional regulatory network (TRN) in Populus trichocarpa (black cottonwood) that has four Secondary wall-associated NAC-Domain1 (PtrSND1) transcription factor (TF) family members as the top-layer regulators. We characterized the function of a MYB (PtrMYB161) TF in this PtrSND1-TRN, using transgenic P trichocarpa cells and whole plants. PtrMYB161 is a third-layer regulator that directly transactivates five wood formation genes. Overexpression of PtrMYB161 in P. trichocarpa (OE-PtrMYB161) led to reduced wood, altered cell type proportions, and inhibited growth. Integrative analysis of wood cell-based chromatin-binding assays with OE-PtrMYB161 transcriptomics revealed a feedback regulation system in the PtrSND1-TRN, where PtrMYB161 represses all four top-layer regulators and one second-layer regulator, PtrMYB021, possibly affecting many downstream TFs in, and likely beyond, the TRN, to generate the observed phenotypic changes. Our data also suggested that the PtrMYB161's repressor function operates through interaction of the base PtrMYB161 target-binding system with gene-silencing cofactors. PtrMYB161 protein does not contain any known negative regulatory domains. CRISPR-based mutants of PtrMYB161 in P. trichocarpa exhibited phenotypes similar to the wild type, suggesting that PtrMYB161's activator functions are redundant among many TFs. Our work demonstrated that PtrMYB161 binds to multiple sets of target genes, a feature that allows it to function as an activator as well as a repressor. The balance of the two functions may be important to the establishment of regulatory homeostasis for normal growth and development.

Modeling cross-regulatory influences on monolignol transcripts and proteins under single and combinatorial gene knockdowns in Populus trichocarpa.

Accurate manipulation of metabolites in monolignol biosynthesis is a key step for controlling lignin content, structure, and other wood properties important to the bioenergy and biomaterial industries. A crucial component of this strategy is predicting how single and combinatorial knockdowns of monolignol specific gene transcripts influence the abundance of monolignol proteins, which are the driving mechanisms of monolignol biosynthesis. Computational models have been developed to estimate protein abundances from transcript perturbations of monolignol specific genes. The accuracy of these models, however, is hindered by their inability to capture indirect regulatory influences on other pathway genes. Here, we examine the manifestation of these indirect influences on transgenic transcript and protein abundances, identifying putative indirect regulatory influences that occur when one or more specific monolignol pathway genes are perturbed. We created a computational model using sparse maximum likelihood to estimate the resulting monolignol transcript and protein abundances in transgenic Populus trichocarpa based on targeted knockdowns of specific monolignol genes. Using in-silico simulations of this model and root mean square error, we showed that our model more accurately estimated transcript and protein abundances, in comparison to previous models, when individual and families of monolignol genes were perturbed. We leveraged insight from the inferred network structure obtained from our model to identify potential genes, including PtrHCT, PtrCAD, and Ptr4CL, involved in post-transcriptional and/or post-translational regulation. Our model provides a useful computational tool for exploring the cascaded impact of single and combinatorial modifications of monolignol specific genes on lignin and other wood properties.

Use of the lignocellulose-degrading bacterium Caldicellulosiruptor bescii to assess recalcitrance and conversion of wild-type and transgenic poplar.

BACKGROUND: Biological conversion of lignocellulosic biomass is significantly hindered by feedstock recalcitrance, which is typically assessed through an enzymatic digestion assay, often preceded by a thermal and/or chemical pretreatment. Here, we assay 17 lines of unpretreated transgenic black cottonwood (Populus trichocarpa) utilizing a lignocellulose-degrading, metabolically engineered bacterium, Caldicellulosiruptor bescii. The poplar lines were assessed by incubation with an engineered C. bescii strain that solubilized and converted the hexose and pentose carbohydrates to ethanol and acetate. The resulting fermentation titer and biomass solubilization were then utilized as a measure of biomass recalcitrance and compared to data previously reported on the transgenic poplar samples. RESULTS: Of the 17 transgenic poplar lines examined with C. bescii, a wide variation in solubilization and fermentation titer was observed. While the wild type poplar control demonstrated relatively high recalcitrance with a total solubilization of only 20% and a fermentation titer of 7.3 mM, the transgenic lines resulted in solubilization ranging from 15 to 79% and fermentation titers from 6.8 to 29.6 mM. Additionally, a strong inverse correlation (R 2 = 0.8) between conversion efficiency and lignin content was observed with lower lignin samples more easily converted and solubilized by C. bescii. CONCLUSIONS: Feedstock recalcitrance can be significantly reduced with transgenic plants, but finding the correct modification may require a large sample set to identify the most advantageous genetic modifications for the feedstock. Utilizing C. bescii as a screening assay for recalcitrance, poplar lines with down-regulation of coumarate 3-hydroxylase 3 (C3H3) resulted in the highest degrees of solubilization and conversion by C. bescii. One such line, with a growth phenotype similar to the wild-type, generated more than three times the fermentation products of the wild-type poplar control, suggesting that excellent digestibility can be achieved without compromising fitness of the tree.