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

Lignocellulosic biomass from plant cell walls is a major source of terrestrial biomass and a potential feedstock for generating fermentable sugars for biofuels and bio-based chemicals. Traditional genetic strategies to improve deconstruction efficiency often target structural genes in cell wall polymer biosynthesis, particularly lignin, but low-lignin plants frequently suffer from growth defects, reduced biomass, altered metabolite profiles, and ectopic defense activation. Attempts to mitigate growth penalties (e.g., suppressor screens, blocking defense signaling, tissue-specific restoration of lignification, haploinsufficiency via editing) still tend to incur yield costs. Techno-economic analyses indicate that biomass yield is the dominant factor in bioenergy crop economics, underscoring the need for approaches that simultaneously reduce recalcitrance and increase yield by leveraging regulators that pleiotropically affect secondary growth. MicroRNAs are key post-transcriptional regulators of plant development and stress responses; miR408 is a conserved 21-nt miRNA whose overexpression increases biomass and seed yield in Arabidopsis and rice, potentially via effects on copper-containing proteins including plantacyanin and laccases. Given laccases’ role in lignification, manipulating miR408 offers a route to decouple growth penalties from reduced recalcitrance. This study tests the hypothesis that Pag-miR408 overexpression in poplar can target specific laccases to delay lignification, increase wall accessibility, improve saccharification without pretreatment, and enhance growth under greenhouse and field conditions, with validation through CRISPR/Cas9 knockout of the laccase targets.

Prior work established that modifying lignin biosynthesis can improve saccharification but often reduces growth and yield. Strategies to alleviate growth penalties include suppressor mutations, defense pathway modulation, tissue-specific complementation of lignification, and gene-editing-based dosage reduction, yet residual yield penalties remain. Analyses emphasize yield as the critical economic driver for bioenergy crops like poplar. miRNAs have been implicated in regulating secondary wall formation and lignification; miR408 overexpression enhances growth and yield in multiple species, with putative targets including laccases. Laccases, together with peroxidases, polymerize monolignols during secondary wall formation. Reduced lignin polymer size and altered composition/distribution correlate with improved extractability and lower recalcitrance. This background motivates testing miR408-mediated laccase regulation to achieve concurrent growth promotion and reduced recalcitrance in poplar.

• Plant materials: Hybrid poplar '84K' (Populus alba × P. glandulosa). Plants grown in phytotron (16 h light/8 h dark, 22°C) for 3, 6, and 12 months; field trial at Beijing Forestry University for ~1 year for selected lines.
• Genetic constructs and transformation: Pag-pre-miR408 cloned into pCAMBIA2300 under CaMV35S for overexpression (miR408_OX). CRISPR/Cas9 constructs with four sgRNAs flanking miR408 to create knockouts (miR408_cr). For targets, LAC19, LAC25, LAC32 overexpression constructs under 35S and CRISPR/Cas9 gene knockouts generated (sgRNAs designed to simultaneously target LAC19, LAC25, LAC32; producing single, double, and triple mutants). Agrobacterium tumefaciens GV3101 leaf-disc transformation used. A CCR2-RNAi line served as positive control for enhanced saccharification.
• Expression analyses: qRT-PCR for miR408 and target transcripts across tissues; promoter-GUS (2 kb Pag-miR408 promoter) to localize expression. RNA-seq on differentiating stem xylem (Hisat2 mapping to P. trichocarpa v3.1; FPKM quantification); KEGG enrichment analyses. Laccase protein levels measured by ELISA.
• Target validation: psRNATarget prediction; dual-luciferase effector-reporter assays in Nicotiana benthamiana testing miR408 regulation of LAC19/25/32 and mutated (non-targetable) controls; 5′ RACE to map miR408-guided cleavage sites in LAC19, LAC25, LAC32; fluorescence in situ hybridization (FISH) co-localization of miR408, LAC19, LAC25 in stem tissues.
• Anatomy and microscopy: Semi-thin sections (toluidine blue staining) for cambium/xylem morphology; TEM for cell wall ultrastructure; phloroglucinol-HCl (Wiesner) staining for lignin deposition across internodes; Image-Pro Plus for quantification. Confocal Raman microspectroscopy (CRM) and stimulated Raman scattering (SRS) imaging to map lignin distribution and quantify intensity across cell walls in specific internodes.
• Cell wall accessibility assays: Binding of GFP-tagged carbohydrate-binding modules recognizing crystalline cellulose (TrCBM1-GFP from T. reesei CBH I; CrCBM3-GFP from C. thermocellum CipA) on hand-cut transverse sections of tissue-cultured and one-year-old stems; fluorescence microscopy with UV lignin autofluorescence for anti-correlation. Alexa Fluor 488-labeled commercial cellulase binding on sections to assess enzyme access kinetics/intensity.
• Saccharification assays: Cell wall residues (methanol and chloroform:methanol extracted) digested with cellulase cocktail without acid pretreatment (72 h, 50°C); sugars quantified by phenol-sulfuric acid; efficiency calculated as released sugars relative to total sugars in CWR determined by two-stage sulfuric acid hydrolysis.
• Cell wall chemistry: Lignin content by acetyl bromide (AcBr) and Klason (acid-insoluble and acid-soluble) methods; structural carbohydrates (monosaccharides) via NREL protocols (GC-FID after derivatization for AcBr assay; standard NREL for Klason runs). Lignin structure by 2D-HSQC NMR on double enzymatic lignin (DEL) isolated after sequential ball-milling and enzyme treatments; S/G ratios and interunit linkage distributions quantified. Lignin molecular weights (Mw, Mn, PDI) measured after acetylation by GPC in THF with polystyrene standards.
• Growth phenotyping: Plant height and basal stem diameter measured in greenhouse and field; internode counts; net photosynthetic rate (NPR). Statistical analyses primarily two-tailed Student’s t-tests; n as specified per experiment.

• Growth promotion in greenhouse: miR408_OX lines (#1, #5, #6) showed increased plant height by 34.75%, 20.42%, 16.94% and basal stem diameter by 27.80%, 15.83%, 11.38% versus WT. Net photosynthetic rate ~40% higher than WT. Knockout lines (miR408_cr #8, #20) showed no significant changes in these parameters.
• Vascular development: miR408_OX plants exhibited a ~42% wider cambial zone, more xylem cells, increased xylem area, more vessels, and enlarged vessel area; fiber cell area unchanged.
• Cell wall accessibility: TrCBM1-GFP and CrCBM3-GFP binding signals were markedly higher in miR408_OX tissues than WT (greater area and intensity), exceeding even CCR2-RNAi in some regions; miR408_cr had very weak signals. Labeled cellulase binding was much stronger in miR408_OX vs WT, indicating enhanced enzyme accessibility.
• Saccharification without pretreatment: One-year-old natural dried stems of miR408_OX had slightly higher total sugars; enzyme-released sugars increased by 110–115% relative to WT. Saccharification efficiency (released/total cell wall sugars) increased by 85–92% across miR408_OX lines.
• Field validation: After one year, field-grown miR408_OX plants had increased height by 42.57%, 41.06%, 38.20% and basal diameter by 35.70%, 33.29%, 31.30% vs WT; TrCBM1-GFP assays confirmed enhanced cell wall accessibility in field samples.
• Lignin deposition and composition: CRM and SRS showed reduced lignin signal intensity in secondary xylem of miR408_OX, with lignin concentrated more in cell corners—consistent with delayed lignification. 2D-HSQC NMR indicated lower S-unit abundance and higher G-unit abundance in miR408_OX; S/G decreased from 2.88 (WT) to 1.98, 2.02, 2.21 in miR408_OX lines. β-O-4 (A) subunits relatively reduced; β–β (B) distribution altered.
• Lignin content and size: AcBr lignin decreased by ~10% vs WT; Klason total lignin decreased by ~4% (both AIL and ASL reduced). Carbohydrate analyses showed little change in glucose (cellulose) but reductions in xylose, mannose, and glucuronic acid (hemicelluloses) in miR408_OX. GPC of acetylated lignin showed decreased Mw (WT 8697 g/mol vs 7313, 7309, 7186 g/mol) and Mn (WT 5033 vs 3967, 4021, 3941 g/mol), with increased PDI (WT 1.73 vs ~1.82–1.84) in miR408_OX.
• Targets of miR408: RNA-seq revealed down-regulation of phenylpropanoid biosynthesis pathway genes in miR408_OX; miR408_cr showed up-regulation of lignin biosynthesis genes (e.g., COMT, CCoAOMT ~2.3×; PAL1/2 and C4H ~2.5–2.8×). psRNATarget predicted LAC19, LAC25, LAC32 as primary targets. qRT-PCR showed reduced transcripts of LAC19/25/32 in miR408_OX. ELISA indicated ~22.5% lower extractable laccase protein in miR408_OX. Dual-LUC assays confirmed repression; 5′ RACE mapped miR408-guided cleavage sites in exon 2 of LAC19 (7/20 clones), LAC25 (6/20), LAC32 (7/20). FISH showed spatial co-localization of miR408 with LAC19/25 in stems, consistent with roles in lignification.
• Genetic validation with laccase mutants: CRISPR triple mutants (lac19 lac25 lac32) and double mutants (lac25 lac32) had significantly increased height and diameter; laccase overexpression lines (LAC19_OX, LAC25_OX, LAC32_OX) had reduced growth. Phloroglucinol staining revealed lighter lignin and looser cell arrangement, with vessel collapse in double/triple mutants; overexpression lines showed stronger staining and WT-like arrangement. Klason lignin increased in laccase overexpression lines by 19.69% (LAC19_OX), 10.67% (LAC25_OX), 14.62% (LAC32_OX). CBM1-GFP binding and labeled cellulase assays showed highest accessibility in lac19 lac25 lac32, consistent with increased saccharification in mutants. CRM confirmed reduced lignin deposition in lac19 lac25 lac32 vessels.

The results demonstrate that miR408 overexpression in poplar can concurrently enhance growth and reduce biomass recalcitrance. By directly targeting three laccases (LAC19, LAC25, LAC32), miR408 delays lignification and modestly lowers lignin content and S/G ratio while decreasing lignin polymer size. These biochemical and structural changes increase cell wall porosity and enzyme accessibility, enabling robust saccharification without acid pretreatment. The observed anatomical changes—wider cambial zone, enlarged xylem cells, increased vessel area—likely contribute to the improved accessibility and may facilitate cell expansion and overall growth. Genetic validation using laccase triple and double mutants recapitulates miR408_OX phenotypes for growth, lignification patterns, and saccharification, confirming laccases as key functional targets. This approach overcomes typical trade-offs seen in lignin-pathway engineering by leveraging post-transcriptional regulation to reduce lignin polymerization rather than dramatically suppressing monolignol biosynthesis, thereby preserving or enhancing growth. The findings support a model in which reduced polymer-polymer cross-linking and delayed lignification increase wall openness and enzyme access during deconstruction while enabling easier microfibril expansion during growth. Further mechanistic studies are needed to fully resolve the coordination between developmental regulation of secondary wall deposition and miR408-mediated biochemical changes.

Overexpression of Pag-miR408 in hybrid poplar enhances biomass yield and saccharification efficiency without the need for acid pretreatment, validated in greenhouse and field conditions. miR408 targets LAC19, LAC25, and LAC32, leading to delayed lignification, reduced lignin content, altered S/G ratios, and decreased lignin polymer sizes, which together increase cell wall accessibility. CRISPR-mediated knockout of these laccases phenocopies the miR408_OX lines, confirming causality. This work demonstrates that manipulating a non-coding RNA can simultaneously promote growth and reduce recalcitrance, offering a promising strategy for bioenergy crop improvement. Future work should dissect the regulatory network linking miR408 to secondary wall formation and evaluate long-term field performance, environmental interactions, and impacts on downstream fermentation yields.

• The reduction in lignin content and compositional changes in miR408_OX are modest; the precise contribution of each change (content, S/G ratio, polymer size, spatial distribution) to saccharification was not deconvoluted.
• While anatomical and biochemical correlations are strong, the developmental coordination and signaling pathways linking miR408 to secondary wall transcriptional networks require further elucidation, as noted by down-regulation of VND7/SND1 and up-regulation of LBD15 in knockouts.
• Saccharification was assessed by sugar release assays; direct fermentation performance and product yields were not reported.
• Long-term, multi-location field trials and assessments of potential ecological or stress-response trade-offs were not included.
• Potential pleiotropic effects of reduced laccase activity in non-xylem tissues were not exhaustively characterized.