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At least 37 records · Page 2
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

PagMYB128 regulates secondary cell wall formation by direct activation of cell wall biosynthetic genes during wood formation in poplar

The biosynthesis of cellulose, lignin, and hemicelluloses in plant secondary cell walls (SCWs) is regulated by a hierarchical transcriptional regulatory network. Here, this network features orthologous transcription factors shared between poplar and Arabidopsis, highlighting a foundational similarity in their genetic regulation. However, knowledge on the discrepant behavior of the transcriptional-level molecular regulatory mechanisms between poplar and Arabidopsis remains limited. In this study, we investigated the function of PagMYB128 during wood formation and found it had broader impacts on SCW formation compared to its Arabidopsis ortholog, AtMYB103. Transgenic poplar trees overexpressing PagMYB128 exhibited significantly enhanced xylem development, with fiber cells and vessels displaying thicker walls, and an increase in the levels of cellulose, lignin, and hemicelluloses in the wood. In contrast, plants with dominant repression of PagMYB128 demonstrated the opposite phenotypes. RNA sequencing and reverse transcription – quantitative polymerase chain reaction showed that PagMYB128 could activate SCW biosynthetic gene expression, and chromatin immunoprecipitation along with yeast one-hybrid, and effector–reporter assays showed this regulation was direct. Further analysis revealed that PagSND1 (SECONDARY WALL-ASSOCIATED NAC-DOMAIN PROTEIN1) directly regulates PagMYB128 but not cell wall metabolic genes, highlighting the pivotal role of PagMYB128 in the SND1-driven regulatory network for wood development, thereby creating a feedforward loop in SCW biosynthesis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Metabolic engineering of p –hydroxybenzoate in poplar lignin

Ester-linked p-hydroxybenzoate occurs naturally in poplar lignin as pendent groups that can be released by mild alkaline hydrolysis. These ‘clip-off’ phenolics can be separated from biomass and upgraded into diverse high-value bioproducts. We introduced a bacterial chorismate pyruvate lyase gene into transgenic poplar trees with the aim of producing more p-hydroxybenzoate from chorismate, itself a metabolic precursor to lignin. By driving heterologous expression specifically in the plastids of cells undergoing secondary wall formation, this strategy achieved a 50% increase in cell-wall-bound p-hydroxybenzoate in mature wood and nearly 10 times more in developing xylem relative to control trees. Comparable amounts also remained as soluble p-hydroxybenzoate-containing xylem metabolites, pointing to even greater engineering potential. Mass spectrometry imaging showed that the elevated p-hydroxybenzoylation was largely restricted to the cell walls of fibres. Finally, transgenic lines outperformed control trees in assays of saccharification potential. Here, this study highlights the biotech potential of cell-wall-bound phenolate esters and demonstrates the importance of substrate supply in lignin engineering.

4-hydroxybenzoic acid↗
DOE OSTI2025

Engineering 2‐Pyrone‐4,6‐Dicarboxylic Acid Production Reveals Metabolic Plasticity of Poplar

Woody biomass is a promising source of fermentable sugars for biofuels and bio-based chemicals, but its industrial use is limited by the costly biorefinery process. A viable strategy to reduce costs involves enhancing both biomass processability and the generation of high-value co-products. Here, we report the implementation of a synthetic metabolic pathway in Populus tremula × P. alba to produce 2-pyrone-4,6-dicarboxylic acid (PDC), a key building block for biodegradable plastics and high-performance materials. This artificial pathway—comprising microbial genes AroG, QsuB, PmdA, PmdB, and PmdC—enabled de novo PDC production in the stems of transgenic poplar. Pathway expression also induced substantial metabolic reprogramming and altered cell wall composition. These include the hyperaccumulation of simple phenolics like protocatechuic acid (PCA) and vanillic acid (VA), alongside reduced levels of p-hydroxybenzoic acid. A large portion of VA was ester-linked to cell wall lignin, while PCA was incorporated into the lignin backbone, forming novel benzodioxane units; concurrently, lignin in transgenic plants exhibited a drastic reduction in guaiacyl- and syringyl-units, with a notable increase in p-hydroxyphenyl-units. Hemicellulose content, particularly xylan, was also significantly increased. Moreover, expression of the PDC-pathway led to the formation of novel VA-derived suberin aromatics, enhancing suberization in bark and roots and improving salt stress tolerance. These changes led to improved saccharification efficiency, with up to 25% more glucose and 2.5 times xylose released from woody biomass. These results demonstrate the metabolic flexibility of poplar and highlight its potential for engineering cost-effective, stress-resilient bioenergy crops with enhanced biorefinery traits.

2-pyrone-4↗
DOE OSTI2026

Diol-enhanced natural deep eutectic solvents for efficient poplar pretreatment

Natural deep eutectic solvents (NDESs) are promising biomass pretreatment media, but their industrial application is often hindered by high viscosity. To address this limitation, diol-enhanced ternary DESs (TDESs) were prepared by incorporating 1,4-butanediol (1,4-BDO) or ethylene glycol (EG) into a choline chloride (ChCl) and 3,4-dihydroxybenzoic acid (DHBA) system. The applied TDESs maintained a liquid state at room temperature and had significantly reduced viscosity compared to the binary DES (BDES). In addition, the applied diols increased lignin solubility and suppressed lignin condensation by intercepting reactive carbocation intermediates. As a result, the recovered lignins from diol-induced TDES pretreatments showed better preservation of β-O-4 linkages and reduced condensation, improving their potential for downstream valorization. The diol-assisted DES systems showed a synergistic effect from the reduced viscosity, enhanced lignin solubility, and suppression of unwanted condensation, resulting in more effective biomass pretreatment performance, including the enhanced delignification and higher enzymatic digestibility compared to BDES. The 1,4-BDO-enhanced DES was also successfully applied to DHBA-enriched transgenic poplar, highlighting its potential for the processing of engineered biomass feedstocks.

3,4-dihydroxybenzoic acid↗
DOE OSTI2021

p HBMT1, a BAHD-family monolignol acyltransferase, mediates lignin acylation in poplar

Abstract Poplar (Populus) lignin is naturally acylated with p-hydroxybenzoate ester moieties. However, the enzyme(s) involved in the biosynthesis of the monolignol–p-hydroxybenzoates have remained largely unknown. Here, we performed an in vitro screen of the Populus trichocarpa BAHD acyltransferase superfamily (116 genes) using a wheatgerm cell-free translation system and found five enzymes capable of producing monolignol–p-hydroxybenzoates. We then compared the transcript abundance of the five corresponding genes with p-hydroxybenzoate concentrations using naturally occurring unrelated genotypes of P. trichocarpa and revealed a positive correlation between the expression of p-hydroxybenzoyl-CoA monolig-nol transferase (pHBMT1, Potri.001G448000) and p-hydroxybenzoate levels. To test whether pHBMT1 is responsible for the biosynthesis of monolignol–p-hydroxybenzoates, we overexpressed pHBMT1 in hybrid poplar (Populus alba × P. grandidentata) (35S::pHBMT1 and C4H::pHBMT1). Using three complementary analytical methods, we showed that there was an increase in soluble monolignol–p-hydroxybenzoates and cell-wall-bound monolignol–p-hydroxybenzoates in the poplar transgenics. As these pendent groups are ester-linked, saponification releases p-hydroxybenzoate, a precursor to parabens that are used in pharmaceuticals and cosmetics. This identified gene could therefore be used to engineer lignocellulosic biomass with increased value for emerging biorefinery strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development

Summary The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships. Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed. MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2‐regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Several root monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis. Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis.

Marqués‐Gálvez, José Eduardo↗
DOE OSTI2025

The effects of expression of a hyperthermophilic endoglucanase and nutrient conditions on poplar growth and physiology

Abstract Efficient production and processing of poplar biomass feedstock requires costly pretreatments and enzyme additives. Transgenic alterations of poplar can reduce the need for these inputs by increasing biomass, improving lignocellulose quality and enhancing nutrient uptake. Previously, a transgenic line of poplar expressing a bacterial hyperthermophilic endoglucanase (TnCelB) in Populus alba × grandidentata (P39) was developed and characterized. This study reports the effects on the TnCelB transgenic poplar line under a reduced nutrient treatment. Overall, the nutrient treatment was the source of more observed significant differences than the genotype. Wild type and TnCelB poplar had similar responses in biomass allocation and net photosynthesis. TnCelB trees had a wrinkled leaf phenotype and relative to wild type, had reduced total biomass, reduced water-use efficiency, and a decreased proportion of cellulose to hemicellulose and lignin. In low nutrient conditions, TnCelB trees had increased structural carbohydrates with stable lignin values. The TnCelB line presents a viable option for poplar biomass feedstock, offering biomass comparable to wild type poplar and more efficient processing, with only mild negative phenotypes.

Natalie, Bethanie M. (ORCID:0009000843856585)↗
DOE OSTI2023

Overexpression of a gibberellin 20-oxidase gene in poplar xylem led to an increase in the size of nanocellulose fibrils and improved paper properties

Cellulose, the major component of secondary cell walls, is the most abundant renewable long-chain polymer on earth. Nanocellulose has become a prominent nano-reinforcement agent for polymer matrices in various industries. We report the generation of transgenic hybrid poplar overexpressing the Arabidopsis gibberellin 20-oxidase1 gene driven by a xylem-specific promoter to increase gibberellin (GA) biosynthesis in wood. X-ray diffraction (XRD) and sum frequency generation spectroscopic (SFG) analyses showed that cellulose in transgenic trees was less crystalline, but the crystal size was larger. The nanocellulose fibrils prepared from transgenic wood had an increased size compared to those from wild type. When such fibrils were used as a reinforcing agent in sheet paper preparation, the mechanical strength of the paper was significantly enhanced. Engineering the GA pathway can therefore affect nanocellulose properties, providing a new strategy for expanding nanocellulose applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Fungal Pretreatment and Enzymatic Hydrolysis of Genetically-Modified Populus trichocarpa

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.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genetically engineered poplar wood effectively enhances the efficiency of deep eutectic solvent-mediated one-pot processing

Although lignocellulosic biomass is a renewable resource with the potential to replace fossil-derived fuels and chemicals, its recalcitrance, largely due to lignin, limits its utilization. Recent advancements in genetic engineering have produced transgenic trees with reduced lignin content and/or modified lignin structure without compromising growth traits. Here, three engineered poplar varieties are evaluated as feedstocks using a biocompatible one-pot deep eutectic solvent-mediated process that integrates biomass fractionation and enzymatic saccharification within a single reactor, eliminating water washing and reconditioning. All transgenic poplars exhibit higher fermentable sugar yields than wild-type (WT) trees. Notably, QsuB poplar, incorporating 3,4-dihydroxybenzoate in lignin, achieves the highest glucose conversion yield of 91.3% (vs. 73.0% from WT). AT5 and MdCHS3 poplars, incorporating ferulate esters and naringenin, also demonstrate improved glucose yields (86.7 and 84.7%, respectively), confirming reduced biomass recalcitrance. Additionally, residual lignins are valorized via hydrogenolysis into phenolic compounds, with comparable alkylphenol production across all lines. These findings demonstrate that the transgenic poplar lines not only serve as superior feedstocks for sugar conversion but also provide a rich resource for phenolic compound production, enhancing the operational and economic viability of integrated biorefinery processes.

biomasses↗
DOE OSTI2022

Engineering Curcumin Biosynthesis in Poplar Affects Lignification and Biomass Yield

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars mainly due to the presence of lignin. By engineering plants to partially replace traditional lignin monomers with alternative ones, lignin degradability and extractability can be enhanced. Previously, the alternative monomer curcumin has been successfully produced and incorporated into lignified cell walls of Arabidopsis by the heterologous expression of DIKETIDE-CoA SYNTHASE (DCS) and CURCUMIN SYNTHASE2 (CURS2). The resulting transgenic plants did not suffer from yield penalties and had an increased saccharification yield after alkaline pretreatment. Here, we translated this strategy into the bio-energy crop poplar. Via the heterologous expression of DCS and CURS2 under the control of the secondary cell wall CELLULOSE SYNTHASE A8-B promoter (ProCesA8-B), curcumin was also produced and incorporated into the lignified cell walls of poplar. ProCesA8-B:DCS_CURS2 transgenic poplars, however, suffered from shoot-tip necrosis and yield penalties. Compared to that of the wild-type (WT), the wood of transgenic poplars had 21% less cellulose, 28% more matrix polysaccharides, 23% more lignin and a significantly altered lignin composition. More specifically, ProCesA8-B:DCS_CURS2 lignin had a reduced syringyl/guaiacyl unit (S/G) ratio, an increased frequency of p-hydroxyphenyl (H) units, a decreased frequency of p-hydroxybenzoates and a higher fraction of phenylcoumaran units. Without, or with alkaline or hot water pretreatment, the saccharification efficiency of the transgenic lines was equal to that of the WT. These differences in (growth) phenotype illustrate that translational research in crops is essential to assess the value of an engineering strategy for applications. Further fine-tuning of this research strategy (e.g., by using more specific promoters or by translating this strategy to other crops such as maize) might lead to transgenic bio-energy crops with cell walls more amenable to deconstruction without settling in yield.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Biological funneling of phenolics from transgenic plants engineered to express the bacterial 3-dehydroshikimate dehydratase (qsuB) gene

The economic and environmental sustainability of lignocellulosic biomass biorefineries is predicated on generating biofuels and bioproducts from cell-wall polysaccharide and lignin polymers. Historical efforts in plant genetic engineering have focused on the development of strategies that facilitate biomass deconstruction, with more recently efforts including the synthesis of high-value chemicals in planta . One such genetic modification is the expression of the bacterial quinate and shikimate utilization B ( qsuB ) gene that increases the accumulation of protocatechuic acid in lignocellulosic biomass. Herein, we evaluated the effectiveness of an alkaline pretreatment process to extract phenolics directly from wild-type and QsuB-transgenic lines of Arabidopsis, poplar, and sorghum, and then upgrade them to the polyester precursor 2-pyrone-4,6-dicarboxylic acid (PDC) with an engineered strain of Novosphingobium aromaticivorans . Protocatechuic acid extracted from all QsuB transgenic lines was found to be mostly in the glycosylated form. Glycosylated protocatechuic acid and other plant-derived phenolics were effectively metabolized by N. aromaticivorans, and PDC production was greatest using extracts from an Arabidopsis QsuB transgenic line (∼5% w/w), followed by QsuB sorghum (∼1.1% w/w), and QsuB poplar (∼0.4% w/w) lines. The comparison of PDC production from wild-type and QsuB transgenic lines of Arabidopsis, poplar, and sorghum demonstrates the utility of a mild alkaline pretreatment to liberate phenolics from plant biomass that are either naturally present or that accumulate as a consequence of genetic engineering strategies. All QsuB transgenic lines outperformed their wild-type counterparts with respect to observed PDC yields. In addition, microbial funneling to PDC was effective even when most of the protocatechuic acid extracted was in glycosylated form, clearly demonstrating that this bacterium can metabolize these aromatic conjugates. These findings illustrate the benefits of combining plant and microbial engineering for bioproduct formation from phenolics in lignocellulosic biorefineries.

Umana, German E.↗
DOE OSTI2025

Crash–and–dash: a new era in tree genome editing

In less than a decade since the first demonstrations of CRISPR genome editing of agronomic genes in several tree species (Zhou et al., 2015; Jia et al., 2016; Ren et al., 2016), this disruptive technology has been deployed for a growing number of traits in both basic and applied research. The precision and efficiency of CRISPR editing allow for the recovery of null mutants in the first generation (Zhou et al., 2015; Elorriaga et al., 2018; Muhr et al., 2018), which is a significant benefit for perennial trees with long generation times. The stability of editing outcomes over multiple years or clonal propagation cycles (Bewg et al., 2022; Chen et al., 2023; Goralogia et al., 2024) is another key advantage since most woody perennials are vegetatively propagated in commercial operations. However, in many countries, gene-edited trees with stably integrated T-DNA face the same regulatory hurdles as traditional transgenics, slowing field trial characterization and the integration of transgenesis with conventional breeding (Boerjan & Strauss, 2024). In an article recently published in New Phytologist, Hoengenaert et al. (2025; doi: 10.1111/nph.20415) demonstrate transgene-free editing in poplar (Populus tremula × alba) that shows promise for wide adoption. The CRISPR-edited, transgene-free canker-resistant citrus (Citrus sinensis) trees (Su et al., 2023) have recently been approved by USDA-APHIS and are exempt from regulation by the US Environmental Protection Agency (EPA) for commercial production. The work by Hoengenaert et al. (2025) suggests a similar path could be followed for purpose-grown plantations for bioenergy, bioproducts, and biomaterials.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The WUSCHELa ( PtoWUSa ) is Involved in Developmental Plasticity of Adventitious Root in Poplar

WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors play critical roles in cell fate determination during plant development. As the founding member of the WOX family, WUSCHEL (WUS) is characterized for its role in maintaining stem cell in meristem. In this study, we investigated the function of Populus tomentosa WUSCHELa (PtoWUSa) in adventitious roots (ARs) in poplar. Expression profile analysis showed that PtoWUSa was not only expressed in shoot apical meristem and stem, but also expressed in ARs. Ectopic expression of PtoWUSa in Arabidopsis resulted in shortened primary root, as well as agravitropism and multiple branches. Overexpression of PtoWUSa in poplar increased the number of ARs but decreased their length. Moreover, the AR tip and lateral root tip became larger and swollen. In addition, the expression of auxin transporter genes PIN-FORMED were downregulated in ARs of transgenic plant. Taken together, these results suggest that PtoWUSa could be involved in AR development in poplar through regulating the polar auxin transport in ARs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Genome resequencing reveals demographic history and genetic architecture of seed salinity tolerance in Populus euphratica

Populus euphratica is a dominant tree species in desert riparian forests and possesses extraordinary adaptation to salinity stress. Exploration of its genomic variation and molecular underpinning of salinity tolerance is important for elucidating population evolution and identifying stress-related genes. Here, we identify approximately 3.15 million single nucleotide polymorphisms using whole-genome resequencing. The natural populations of P. euphratica in northwest China are divided into four distinct clades that exhibit strong geographical distribution patterns. Pleistocene climatic fluctuations and tectonic deformation jointly shaped the extant genetic patterns. A seed germination rate-based salinity tolerance index was used to evaluate seed salinity tolerance of P. euphratica and a genome-wide association study was implemented. A total of 38 single nucleotide polymorphisms were associated with seed salinity tolerance and were located within or near 82 genes. Expression profiles showed that most of these genes were regulated under salt stress, revealing the genetic complexity of seed salinity tolerance. Furthermore, DEAD-box ATP-dependent RNA helicase 57 and one undescribed gene (CCG029559) were demonstrated to improve the seed salinity tolerance in transgenic Arabidopsis. These results provide new insights into the demographic history and genetic architecture of seed salinity tolerance in desert poplar.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Evaluation of engineered low-lignin poplar for conversion into advanced bioproducts

Lignocellulosic resources are promising feedstocks for the manufacture of bio-based products and bioenergy. However, the inherent recalcitrance of biomass to conversion into simple sugars currently hinders the deployment of advanced bioproducts at large scale. Lignin is a primary contributor to biomass recalcitrance as it protects cell wall polysaccharides from degradation and can inhibit hydrolytic enzymes via non-productive adsorption. Several engineering strategies have been designed to reduce lignin or modify its monomeric composition. For example, expression of bacterial 3-dehydroshikimate dehydratase (QsuB) in poplar trees resulted in a reduction in lignin due to redirection of metabolic flux toward 3,4-dihydroxybenzoate at the expense of lignin. This reduction was accompanied with remarkable changes in the pools of aromatic compounds that accumulate in the biomass. The impact of these modifications on downstream biomass deconstruction and conversion into advanced bioproducts was evaluated in the current study. Using ionic liquid pretreatment followed by enzymatic saccharification, biomass from engineered trees released more glucose and xylose compared to wild-type control trees under optimum conditions. Fermentation of the resulting hydrolysates using Rhodosporidium toruloides strains engineered to produce α-bisabolene, epi-isozizaene, and fatty alcohols showed no negative impact on cell growth and yielded higher titers of bioproducts (as much as + 58%) in the case of QsuB transgenics trees. Our data show that low-recalcitrant poplar biomass obtained with the QsuB technology has the potential to improve the production of advanced bioproducts.

09 BIOMASS FUELS↗
DOE OSTI2021

Exogenous chalcone synthase expression in developing poplar xylem incorporates naringenin into lignins

Abstract Lignin, a polyphenolic polymer, is a major chemical constituent of the cell walls of terrestrial plants. The biosynthesis of lignin is a highly plastic process, as highlighted by an increasing number of noncanonical monomers that have been successfully identified in an array of plants. Here, we engineered hybrid poplar (Populus alba x grandidentata) to express chalcone synthase 3 (MdCHS3) derived from apple (Malus domestica) in lignifying xylem. Transgenic trees displayed an accumulation of the flavonoid naringenin in xylem methanolic extracts not inherently observed in wild-type trees. Nuclear magnetic resonance analysis revealed the presence of naringenin in the extract-free, cellulase-treated xylem lignin of MdCHS3-poplar, indicating the incorporation of this flavonoid-derived compound into poplar secondary cell wall lignins. The transgenic trees also displayed lower total cell wall lignin content and increased cell wall carbohydrate content and performed significantly better in limited saccharification assays than their wild-type counterparts.

59 BASIC BIOLOGICAL SCIENCES↗