Transcription factor PagMYB31 positively regulates cambium activity and negatively regulates xylem development in poplar.

Wood formation involves consecutive developmental steps, including cell division of vascular cambium, xylem cell expansion, secondary cell wall (SCW) deposition, and programmed cell death. In this study, we identified PagMYB31 as a coordinator regulating these processes in Populus alba × Populus glandulosa and built a PagMYB31-mediated transcriptional regulatory network. PagMYB31 mutation caused fewer layers of cambial cells, larger fusiform initials, ray initials, vessels, fiber and ray cells, and enhanced xylem cell SCW thickening, showing that PagMYB31 positively regulates cambial cell proliferation and negatively regulates xylem cell expansion and SCW biosynthesis. PagMYB31 repressed xylem cell expansion and SCW thickening through directly inhibiting wall-modifying enzyme genes and the transcription factor genes that activate the whole SCW biosynthetic program, respectively. In cambium, PagMYB31 could promote cambial activity through TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)/PHLOEM INTERCALATED WITH XYLEM (PXY) signaling by directly regulating CLAVATA3/ESR-RELATED (CLE) genes, and it could also directly activate WUSCHEL HOMEOBOX RELATED4 (PagWOX4), forming a feedforward regulation. We also observed that PagMYB31 could either promote cell proliferation through the MYB31-MYB72-WOX4 module or inhibit cambial activity through the MYB31-MYB72-VASCULAR CAMBIUM-RELATED MADS2 (VCM2)/PIN-FORMED5 (PIN5) modules, suggesting its role in maintaining the homeostasis of vascular cambium. PagMYB31 could be a potential target to manipulate different developmental stages of wood formation.

Root-specific NF-Y family transcription factor, PdNF-YB21, positively regulates root growth and drought resistance by abscisic acid-mediated indoylacetic acid transport in Populus.

Root growth control plays an important role in plant adaptation to drought stress, but the underlying molecular mechanisms of this control remain largely elusive. Here, a root-specific nuclear factor Y (NF-Y) transcription factor PdNF-YB21 was isolated from Populus. The functional mechanism of PdNF-YB21 was characterised by various morphological, physiological, molecular, biochemical and spectroscopy techniques. Overexpression of PdNF-YB21 in poplar promoted root growth with highly lignified and enlarged xylem vessels, resulting in increased drought resistance. By contrast, CRISPR/Cas9-mediated poplar mutant nf-yb21 exhibited reduced root growth and drought resistance. PdNF-YB21 interacted with PdFUSCA3 (PdFUS3), a B3 domain transcription factor. PdFUS3 directly activated the promoter of the abscisic acid (ABA) synthesis key gene PdNCED3, resulting in a significant increase in root ABA content in poplars subjected to water deficit. Coexpression of poplar NF-YB21 and FUS3 significantly enhanced the expression of PdNCED3. Furthermore, ABA promoted indoylacetic acid transport in root tips, which ultimately increased root growth and drought resistance. Taken together, our data indicate that NF-YB21-FUS3-NCED3 functions as an important avenue in auxin-regulated poplar root growth in response to drought.

MicroRNA857 Is Involved in the Regulation of Secondary Growth of Vascular Tissues in Arabidopsis.

MicroRNAs (miRNAs) are endogenous small RNAs that repress target gene expression posttranscriptionally, and are critically involved in various developmental processes and responses to environmental stresses in eukaryotes. MiRNA857 is not widely distributed in plants and is encoded by a single gene, AtMIR857, in Arabidopsis (Arabidopsis thaliana). The functions of miR857 and its mechanisms in regulating plant growth and development are still unclear. Here, by means of genetic analysis coupled with cytological studies, we investigated the expression pattern and regulation mechanism of miR857 and its biological functions in Arabidopsis development. We found that miR857 regulates its target gene, Arabidopsis LACCASE7, at the transcriptional level, thereby reducing laccase activity. Using stimulated Raman scattering and x-ray microtomography three-dimensional analyses, we showed that miR857 was involved in the regulation of lignin content and consequently morphogenesis of the secondary xylem. In addition, miR857 was activated by SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 in response to low copper conditions. Collectively, these findings demonstrate the role of miR857 in the regulation of secondary growth of vascular tissues in Arabidopsis and reveal a unique control mechanism for secondary growth based on the miR857 expression in response to copper deficiency.

Unveiling the Structural Characteristics of Lignin and Lignin-Carbohydrate Complexes in Fibers and Parenchyma Cells of Moso Bamboo during Different Growing Years.

Understanding and recognizing the structural characteristics of lignin-carbohydrate complexes (LCCs) and lignin in different growth stages and tissue types of bamboo will facilitate industrial processes and practical applications of bamboo biomass. Herein, the LCC and lignin samples were sequentially isolated from fibers and parenchyma cells of bamboo with different growth ages. The diverse yields of sequential fractions not only reflect the different biomass recalcitrance between bamboo fibers and parenchyma cells but also uncover the structural heterogeneity of these tissues at different growth stages. The molecular structures and structural inhomogeneities of the isolated lignin and LCC samples were comprehensively investigated. The results showed that the structural features of lignin and LCC linkages in parenchyma cells were abundant in ß-O-4 linkages but less with carbon-carbon linkages, suggesting that lignin and cross-linked LCC in parenchyma cells are simple in nature and easily to be tamed and tractable in the current biorefinery. Parallelly, the different ball-milled samples were directly characterized by high-resolution (800 M) solution-state 2D-HSQC NMR to analyze the whole lignocellulosic material. Overall, the scheme presented in this study will provide a comprehensive understanding of lignin and LCC linkages in fibers and parenchyma cells of bamboo and enable the utilization of bamboo biomass.

Promoting the disassemble and enzymatic saccharification of bamboo shoot shells via efficient hydrated alkaline deep eutectic solvent pretreatment.

Pretreatment is a key process restricting the development of biorefinery. This work developed a pretreatment process based on an ethanolamine/acetamide alkaline deep eutectic solvent (ADES). Under microwave assistance, pure ADES pretreatment at 100 °C for 10 min achieved 95.9 % delignification and 95.2 % hemicellulose removal of bamboo shoot shells (BSS). Further, when 75 % water was added to pure DES to prepare hydrated DES (75 %-HADES), impressive delignification (93.2 %), hemicellulose removal (92.2 %) and cellulose recovery (94.8 %) were still achieved. The cellulose digestibility of the 75 %-HADES pretreated solid residue was significantly increased from 12.2 % (the control) to 91.2 %. Meanwhile, the structural features of hemicellulose and lignin macromolecules fractionated by 75 %-HADES pretreatment were well preserved, offering opportunities for downstream utilization. Overall, this work proposes an effective pretreatment strategy with the potential to enable the utilization of all major components of bamboo shoot shells.

Simultaneous production of furfural, lignin and cellulose-rich residue from Eucalyptus urophylla × E. grandis by ChCl/1,2-propanediol/MIBK biphasic system pretreatment.

A facile biphasic system composed of choline chloride (ChCl)-based deep eutectic solvent (DES) and methyl isobutyl ketone (MIBK) was developed to realize the furfural production, lignin separation and preparation of fermentable glucose from Eucalyptus in one-pot. Results showed that the ChCl/1,2-propanediol/MIBK system owned the best property to convert hemicelluloses into furfural. Under the optimal conditions (MRChCl:1,2-propanediol = 1:2, raw materials:DES:MIBK ratio = 1:4:8 g/g/mL, 0.075 mol/L AlCl3·6H2O, 140 °C, and 90 min), the furfural yield and glucose yield reached 65.0 and 92.2 %, respectively. Meanwhile, the lignin with low molecular weight (1250-1930 g/mol), low polydispersity (DM = 1.25-1.53) and high purity (only 0.08-2.59 % carbohydrate content) was regenerated from the biphasic system. With the increase of pretreatment temperature, the ß-O-4, ß-ß and ß-5 linkages in the regenerated lignin were gradually broken, and the content of phenolic hydroxyl groups increased, but the content of aliphatic hydroxyl groups decreased. This research provides a new strategy for the comprehensive utilization of lignocellulose in biorefinery process.

Lignocellulosic biomass-derived functional nanocellulose for food-related applications: A review.

In recent years, nanocellulose (NC) has gained significant attention due to its remarkable properties, such as adjustable surface chemistry, extraordinary biological properties, low toxicity and low density. This review summarizes the preparation of NC derived from lignocellulosic biomass (LCB), including cellulose nanofibrils (CNF), cellulose nanocrystals (CNC), and lignin-containing cellulose nanofibrils (LCNF). It focuses on examining the impact of non-cellulosic components such as lignin and hemicellulose on the functionality of NC. Additionally, various surface modification strategies of NC were discussed, including esterification, etherification and silylation. The review also emphasizes the progress of NC application in areas such as Pickering emulsions, food packaging materials, food additives, and hydrogels. Finally, the prospects for producing NC from LCB and its application in food-related fields are examined. This work aims to demonstrate the effective benefits of preparing NC from lignocellulosic biomass and its potential application in the food industry.

Multifunctional composite film of curcumin Pickering emulsion stabilized by lignocellulose nanofibrils isolated from bamboo shoot shells for monitoring shrimp freshness.

Concerns about food safety and environmental impact from chemical surfactants have prompted interest in natural lignocellulosic materials as alternatives. In this study, we combined hydrated deep eutectic solvent (DES) pretreatment with ultrasound treatment to prepare lignocellulosic nanofibrils (LCNF) from bamboo shoot shells with appropriate surface properties for stabilizing Pickering emulsions. The pretreatment intensity effectively modulated the surface characteristics of LCNF, achieving desirable wettability through lignin retention and in-situ esterification. The resulting LCNF/curcumin Pickering emulsion (CPE) demonstrated curcumin protection and pH-responsive color changes, while the ensuing CPE/PVA composite film exhibited ultraviolet shielding, mechanical strength, oxygen barrier, and antioxidant properties. Furthermore, the CPE/PVA film showed promise as a real-time indicator for monitoring shrimp freshness, maintaining sensitivity to spoilage even after six months of storage. These findings advance the advancement of green LCNF technologies, providing eco-friendly solutions for valorizing bamboo shoot shells and enhancing the application of LCNF in Pickering emulsions.

Harnessing Renewable Lignocellulosic Potential for Sustainable Wastewater Purification.

Utilizing renewable lignocellulosic resources for wastewater remediation is crucial to achieving sustainable social development. However, the resulting by-products and the synthetic process characterized by complexity, high cost, and environmental pollution limit the further development of lignocellulose-based materials. Here, we developed a sustainable strategy that involved a new functional deep eutectic solvent (DES) to deconstruct industrial xylose residue into cellulose-rich residue with carboxyl groups, lignin with carboxyl and quaternary ammonium salt groups, and DES effluent rich in lignin fragments. Subsequently, these fractions equipped with customized functionality were used to produce efficient wastewater remediation materials in cost-effective and environmentally sound manners, namely, photocatalyst prepared by carboxyl-modified cellulose residue, biochar-based adsorbent originated from modified lignin, and flocculant synthesized by self-catalytic in situ copolymerization of residual DES effluent at room temperature. Under the no-waste principle, this strategy upgraded the whole components of waste lignocellulose into high-value-added wastewater remediation materials with excellent universality. These materials in coordination with each other can stepwise purify high-hazardous mineral processing wastewater into drinkable water, including the removal of 99.81% of suspended solids, almost all various heavy metal ions, and 97.09% chemical oxygen demand, respectively. This work provided promising solutions and blueprints for lignocellulosic resources to alleviate water shortages while also advancing the global goal of carbon neutrality.

Variation pattern in the macromolecular (cellulose, hemicelluloses, lignin) composition of cell walls in Pinus tabulaeformis tree trunks at different ages as revealed using multiple techniques.

The plant cell wall is a complex, heterogeneous structure primarily composed of cellulose, hemicelluloses, and lignin. Exploring the variations in these three macromolecules over time is crucial for understanding wood formation to enhance chemical processing and utilization. Here, we comprehensively analyzed the chemical composition of cell walls in the trunks of Pinus tabulaeformis using multiple techniques. In situ analysis showed that macromolecules accumulated gradually in the cell wall as the plant aged, and the distribution pattern of lignin was opposite that of polysaccharides, and both showed heterogenous distribution patterns. In addition, gel permeation chromatography (GPC) results revealed that the molecular weights of hemicelluloses decreased while that of lignin increased with age. Two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D-HSQC NMR) analysis indicated that hemicelluloses mainly comprised galactoglucomannan and arabinoglucuronoxylan, and the lignin types were mainly comprised guaiacyl (G) and p-hydroxyphenyl (H) units with three main linkage types: ß-O-4, ß-ß, and ß-5. Furthermore, the C-O bond (ß-O-4) signals of lignin decreased while the C-C bonds (ß-ß and ß-5) signals increased over time. Taken together, these findings shed light on wood formation in P. tabulaeformis and lay the foundation for enhancing the processing and use of wood and timber products.

Fractionation and evaluation of light-colored lignin extracted from bamboo shoot shells using hydrated deep eutectic solvents.

In this study, light-colored lignin was extracted from bamboo shoot shells (BSS) using a hydrated deep eutectic solvent (DES) pretreatment. The hydrated DES used in pretreatment consist of formic acid, benzyl triethylammonium chloride (BTEAC) and water. The pretreatment using a hydrated DES containing 30% water (H30) demonstrate efficient delignification (82.9%). Additionally, the hydrated DES protected the ß-O-4 linkage from excessive cleavage and recondensation as well as keep the light-colored of lignin. Moreover, the hydrated DES extracted lignin exhibits superior antioxidant performance and tyrosinase inhibitory capacity compared to the control. Notably, incorporating 5% lignin of H30-extracted lignin into a commercial suncream led to a remarkable enhancement of the SPF value, elevating from 14.8 to 32.6. In summary, the proposed hydrated DES pretreatment method offers significant benefits for extracting light-colored lignin, thereby promoting the multifunctional application of lignin in cosmetics.

Green and sustainable production of high-purity lignin microparticles with well-preserved substructure and enhanced anti-UV/oxidant activity using peroxide-promoted alkaline deep eutectic solvent.

Deep eutectic solvents (DESs) have emerged as promising and eco-friendly solvents for the efficient extraction of lignin from biomass due to their low cost and environmental benefits. Nevertheless, the prevalent use of acidic DESs in lignin extraction often results in excessive depolymerization and recondensation of lignin, thereby impeding its downstream applications. In this study, we developed a range of alkaline DESs (ADESs), both pure and peroxide-containing, for the extraction of high-quality lignin from bamboo. Moreover, carbon dioxide (CO2) was employed for the precipitation and regeneration of the extracted lignin. The obtained lignin fractions were comprehensively characterized in terms of yield, purity, morphology, solubility, structural features, and anti-UV/oxidant activity. The results showed that the monoethanolamine-based ADES demonstrated superior performance among the pure ADESs. Structural analysis confirmed the well-preserved substructures of lignin fractions obtained using ADESs, with ß-O-4 bond retention ranging from 49.8 % to 68.4 %. The incorporation of a suitable amount of peroxide improved lignin yield, morphology, solubility, and anti-UV/oxidant activity. Additionally, the anti-UV/oxidant activity of lignin exhibited a positive correlation with its phenolic hydroxyl content. This study provides a valuable reference for the green and sustainable production and valorization of lignin within the existing biorefinery framework.

Structural elucidation and targeted valorization of untractable lignin from pre-hydrolysis liquor of xylose production via a simple and robust separation approach.

Effective separation of lignin macromolecules from the xylose pre-hydrolysates (XPH) during the xylose production, thus optimizing the separation and purification process of xylose, is of great significance for reducing the production costs, achieving the high value-added utilization of lignin and increasing the industrial revenue. In this study, a simple and robust method (pH adjustment) for the separation of lignin from XPH was proposed and systematically compared with the conventional acid-promoted lignin precipitation method. The results showed that the lignin removal ratio (up to 60.34 %) of this simple method was higher than that of the conventional method, and the proposed method eliminated the necessity of heating and specialized equipment, which greatly reduced the separation cost. Meanwhile, this simple method does not destroy the components in XPH (especially xylose), ensuring the yield of the target product. On the other hand, the obtained lignin was nano-scale with less condensed structures, which also possessed small molecular weights with narrow distribution, excellent antioxidant activity (8-14 times higher than commercial antioxidants) and UV protection properties. In conclusion, the proposed simple separation method could effectively separate lignin from XPH at low cost, and the obtained lignin had potential commercial applications, which would further enhance the overall profitability of industrial production.

Enhancing saccharification of bamboo shoot shells by rapid one-pot pretreatment of hydrated deep eutectic solvent.

In this work, a rapid one-pot hydrated deep eutectic solvent (DES) pretreatment was proposed to facilitate the conversion of carbohydrates from lignocellulosic biomass to monosaccharides. Specifically, the pure and hydrated DES based on benzyl triethylammonium chloride (BTEAC), formic acid (FA) and water was used to pretreat bamboo shoot shells (BSS) by microwave heating. The pretreated solid residues were enzymatically saccharified to produce fermentable sugars, and the hydrolyzed carbohydrates and lignin remained in the hydrolyzate. The results showed that the yield of monosaccharides from the hydrated DES hydrolyzate (193.7-228.4 g/kg) was significantly higher than that (45.9-66.1 g/kg) of pure DES. The 30% hydrated DES pretreatment achieved the best glucose yield (89.03%) and a total monosaccharides yield of 555.4 g/kg, which corresponded to a conversion ratio of carbohydrates to monosaccharides of 87.0%. The proposed process is a robust method for the efficiently convert carbohydrates from BSS into monosaccharides.

Conversion of control and genetically-modified poplar into multi-scale products using integrated pretreatments.

In this work, a green and robust pretreatment which integrated acetic acid-catalyzed hydrothermal and wet mechanical pretreatment, was developed to efficiently produce high yield (up to 40.12%) of xylooligosaccharides and digestible substrates from Caffeoyl Shikimate Esterase down-regulated and control poplar wood. Subsequently, superhigh yield (more than 95%) of glucose and residual lignin were obtained after a moderate enzymatic hydrolysis. The residual lignin fraction exhibited a well-preserved ß-O-4 linkages (42.06/100Ar) and high S/G ratio (6.42). Subsequently, lignin-derived porous carbon was successfully synthesized, and it exhibited a high specific capacitance of 273.8 F g-1 at 1.0 A g-1 and long cycling stability (remained 98.5% after 10,000 cycles at 5.0 A g-1) compared to control poplar wood, demonstrating that special advantage of this genetically-modified poplar in this integrated process. This work developed an energy-saving and eco-friendly pretreatment technology as a waste-free route for converting different lignocellulosic biomass to multiple products.

Molecular insights into the evolution of woody plant decay in the gut of termites.

Plant cell walls represent the most abundant pool of organic carbon in terrestrial ecosystems but are highly recalcitrant to utilization by microbes and herbivores owing to the physical and chemical barrier provided by lignin biopolymers. Termites are a paradigmatic example of an organism's having evolved the ability to substantially degrade lignified woody plants, yet atomic-scale characterization of lignin depolymerization by termites remains elusive. We report that the phylogenetically derived termite Nasutitermes sp. efficiently degrades lignin via substantial depletion of major interunit linkages and methoxyls by combining isotope-labeled feeding experiments and solution-state and solid-state nuclear magnetic resonance spectroscopy. Exploring the evolutionary origin of lignin depolymerization in termites, we reveal that the early-diverging woodroach Cryptocercus darwini has limited capability in degrading lignocellulose, leaving most polysaccharides intact. Conversely, the phylogenetically basal lineages of "lower" termites are able to disrupt the lignin-polysaccharide inter- and intramolecular bonding while leaving lignin largely intact. These findings advance knowledge on the elusive but efficient delignification in natural systems with implications for next-generation ligninolytic agents.

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.

Manipulating microRNA miR408 enhances both biomass yield and saccharification efficiency in poplar.

The conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis, with associated negative growth impacts. Here we demonstrate, for both laboratory- and field-grown plants, that expression of Pag-miR408 in poplar (Populus alba × P. glandulosa) significantly enhances saccharification, with no requirement for acid-pretreatment, while promoting plant growth. The overexpression plants show increased accessibility of cell walls to cellulase and scaffoldin cellulose-binding modules. Conversely, Pag-miR408 loss-of-function poplar shows decreased cell wall accessibility. Overexpression of Pag-miR408 targets three Pag-LACCASES, delays lignification, and modestly reduces lignin content, S/G ratio and degree of lignin polymerization. Meanwhile, the LACCASE loss of function mutants exhibit significantly increased growth and cell wall accessibility in xylem. Our study shows how Pag-miR408 regulates lignification and secondary growth, and suggest an effective approach towards enhancing biomass yield and saccharification efficiency in a major bioenergy crop.

Efficient fractionation of bamboo residue by autohydrolysis and deep eutectic solvents pretreatment.

Bamboo processing residue, which is rich in parenchyma cells, was treated as huge waste in bamboo processing industry, such as reassemble bamboo and bamboo flooring. Herein, autohydrolysis and rapid different deep eutectic solvents (DES) delignification strategy were consecutively performed to remove hemicelluloses and lignin from bamboo processing residue. The xylooligosaccharides (XOS) with high yield (34.35%) was achieved in the autohydrolysis process. Results showed that alkaline DES pretreatment resulted in the highest glucose yield (88.22%) and relatively high delignification rate (83.75%) as well as well-preserved lignin structures. However, the lignin fractions obtained under acidic DES conditions were tending to assemble into lignin nanoparticles (LNPs) and having excellent antioxidant activity as compared to those obtained from alkaline DES system. In brief, the combination of autohydrolysis and rapid DES delignification can achieve orientated fractionation of the components from the industrialized bamboo.

Structural elucidation and targeted valorization of poplar lignin from the synergistic hydrothermal-deep eutectic solvent pretreatment.

Elucidating the structural variations of lignin during the pretreatment is very important for lignin valorization. Herein, poplar wood was pretreated with an integrated process, which was composed of AlCl3-catalyzed hydrothermal pretreatment (HTP, 130-150 °C, 1.0 h) and mild deep-eutectic solvents (DES, 100 °C, 10 min) delignification for recycling lignin fractions. Confocal Raman Microscopy (CRM) was developed to visually monitor the delignification process during the HTP-DES pretreatment. NMR characterizations (2D-HSQC and 31P NMR) and elemental analysis demonstrated that the lignin fractions had undergone the following structural changes, such as dehydration, depolymerization, condensation. Molecular weights (GPC), microstructure (SEM and TEM), and antioxidant activity (DPPH analysis) of the lignins revealed that the DES delignification resulted in homogeneous lignin fragments (1.32 < PDI < 1.58) and facilitated the rapid assemblage of lignin nanoparticles (LNPs) with controllable nanoscale sizes (30-210 nm) and excellent antioxidant activity. These findings will enhance the understanding of structural transformations of the lignin during the integrated process and maximize the lignin valorization in a current biorefinery process.