Xylem cells are surrounded by primary and secondary cell walls. Formation of primary walls is regulated by the cell wall integrity surveillance system, but it is unclear if the deposition of secondary walls is similarly regulated. To study this question, we introduced to aspen three different enzymes cleaving cell wall-localized xylan and we suppressed xylan synthase components either ubiquitously or specifically during secondary wall formation using the Populus trichocarpa GT43B promoter. When xylan was ubiquitously altered, 95% of lines showed reduced growth, whereas when it was altered during secondary wall deposition, 30% of lines grew better, with the rest having no growth impairment, suggesting opposite effects of primary and secondary wall disturbances. To detect the mechanism of growth stimulation by disturbed deposition of the secondary wall, we analyzed changes in wood quality traits, chemistry, transcriptomics, metabolomics and hormonomics in transgenic lines. We found increased tension wood production, reduced S- and H-lignin, and changes in several metabolites in common in these lines. Remorin REM1.3 and NRL2 (NPH3 family) transcripts increased, and changes in jasmonates, abscisic acid, and salicylic acid occurred in secondary wall-forming xylem, suggesting their involvement in secondary wall integrity surveyance and signaling. The data indicate that a unique program mediates responses to secondary wall impairment that induces growth.

Wood is the most abundant renewable natural resource composed of different polysaccharides and lignin, but its utilisation is hampered by intermolecular linkages between these components forming lignin-carbohydrate complexes (LCCs) causing recalcitrance. The links between glucuronoxylan and the γ-C of lignin (γ-ester linkages) are thought to contribute to one-third of LCCs, but direct evidence for their natural occurrence and their role in recalcitrance has been scarce so far. To address these issues, Phanerochaete carnosa glucuronoyl esterase (PcGCE), hydrolysing γ-ester linkages, was expressed in cell walls of developing wood in hybrid aspen (Populus tremula L. × tremuloides Michx.). The enzyme reduced HSQC 2D NMR signals corresponding to the γ-esters and xylan in dioxane-extracted LCCs without altering glucuronoxylan content or structure. This increased acid solubility of lignin and decreased lignin content. Reduced wood recalcitrance was shown by increased sugar yields and glucose production rates (by approx. 20%) in saccharification without pretreatment and increased xylan extractability by subcritical water (by approx. 70%). Moreover, trees expressing PcGCE exhibited greater primary and secondary growth. Transcriptomics and metabolomics analyses in developing wood suggested that growth could have been induced by a higher transcription of SMR2 and RPOTmp, which was likely triggered by the secondary cell wall integrity signalling. The results provide evidence for the natural existence of LCC γ-esters and their significant contribution to lignocellulose recalcitrance. Furthermore, they show that reducing γ-ester linkages could increase plant productivity.

The evaluation of genomic selection (GS) efficiency in forestry has primarily relied on cross-validation schemes that split the same population within a single generation for both training and validation. While useful, this approach may not be reliable for multigenerational breeding. To our knowledge, this is the first study to assess genomic prediction in Norway spruce using a large dataset spanning two generations in two environments. We trained pedigree-based (ABLUP) and marker-based (GBLUP) prediction models under three approaches: forward prediction, backward prediction, and across-environment prediction. The models were evaluated for ring-width, solid-wood and tracheid characteristics, using 6,000 phenotyped and 2,500 genotyped individual. Predictive ability (PA) and prediction accuracy (ACC) were estimated using an independent validation method, ensuring no individuals were shared between training and validation datasets. To assess the trade-off between comprehensive radial history and practical direct methods, we compared GBLUP models trained with cumulative area-weighted density (AWE-GBLUP) and single annual-ring density (SAD-GBLUP) from mother plus-trees. These models were validated using early and mature-stage progeny density measurements across two trials. Despite the smaller number of individuals used in the GBLUP models, both PA and ACC were generally comparable to those of the ABLUP model, particularly for cross-environment predictions. Overall, forward and backward predictions were significantly higher for density-related and tracheid properties, suggesting that across-generation predictions are feasible for wood properties but may be challenging for growth and low-heritability traits. Notably, SAD-GBLUP provided comparable prediction accuracies to AWE-GBLUP, supporting the use of more practical and cost-effective phenotyping methods in operational breeding programs. Our findings highlight the need for context-specific models to improve the accuracy and reliability of genomic prediction in forest tree breeding. Future efforts might aim to expand training populations, incorporate non-additive genetic effects, and validate model performance across cambial ages while accounting for climatic variability during the corresponding growth years. Overall, this study offers a valuable foundation for implementing GS in Norway spruce breeding programs.

Spatial information on wood structure and chemistry is crucial for understanding wood functionality. We present a high-throughput and high-resolution near-infrared (NIR) method for combined imaging of the physical and chemical properties of stem sections from Populus trees. Pyrolysis-GC/MS data was used for sensitive and spatially resolved calibration of wood chemistry while SilviScan'" analyses provided reference data for wood physical properties with 25 mu m resolution for wood density and 0.2-2.0 mm for microfibril angle (MFA). NIR prediction models were trained and calibrated on material from both fieldand greenhouse-grown trees. Thus, the method was developed for NIR imaging of stem samples as small as 4 mm in diameter with an image resolution of 0.03 mm for small-diameter samples and 0.5 mm for samples with multiple annual rings. The NIR model performance, tested against data not used in the training set, reached the coefficient of determination (R2pred) values for wood density and MFA of 0.60 and 0.72, respectively. The NIR models for wood chemistry showed R2pred values of 0.78 and 0.77 for carbohydrates and lignin, respectively. Models for the G-, S- and H-type lignin had R2pred values between 0.58 and 0.86. In addition, we developed a prediction model for the determination of tension wood distribution. According to this model, tension wood was frequently observed in young greenhouse samples, which might explain the higher variation found in the chemical and physical properties of wood in greenhouse-grown compared to field-grown trees. The study also demonstrated that NIR-model estimations in image format can capture spatial variations that are not detectable in bulk analyses of wood properties. Examples of the method applied to greenhouse-grown trees highlight the efforts to develop NIR models with good prediction accuracies based on high-resolution data.

Xylem cells are surrounded by primary and secondary cell walls. Formation of primary walls is regulated by the cell wall integrity surveyance system, but it is unclear if the deposition of secondary walls is similarly regulated. To study this question, we introduced to aspen three different enzymes cleaving cell wall-localized xylan and we suppressed xylan synthase components either ubiquitously or specifically during secondary wall formation. When xylan was ubiquitously altered, 95% of lines showed reduced growth, whereas when it was altered during secondary wall deposition, 30% of lines grew better with the rest having no growth impairment, suggesting opposite effects of primary and secondary wall disturbances. To detect mechanism of growth stimulation by disturbed deposition of secondary wall, we analyzed changes in wood quality traits, chemistry, transcriptomics, metabolomics and hormonomics in transgenic lines. We found increased tension wood production, reduced S- and H-lignin, and changes in several metabolites in common in these lines. Remorin REM1.3 and NRL2 ( NPH3 family) transcripts increased and changes in jasmonates, ABA and SA occurred in secondary wall-forming xylem suggesting their involvement in secondary wall integrity surveyance and signaling. The data indicate that a unique program mediates responses to secondary wall impairment that induces growth. Highlight Disturbance of the deposition of secondary walls that are responsible for the mechanical strength of plant bodies can increase growth with discrete changes in gene expression, hormone levels and metabolism. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:Forest geneticists typically use provenances to account for population differences in their improvement schemes; however, the historical records of the imported materials might not be very precise or well-aligned with the genetic clusters derived from advanced molecular techniques. The main objective of this study was to assess the impact of marker-based population structure on genetic parameter estimates related to growth and wood properties and their trade-offs in Norway spruce, by either incorporating it as a fixed effect (model-A) or excluding it entirely from the analysis (model-B). RESULTS:Our results indicate that models incorporating population structure significantly reduce estimates of additive genetic variance, resulting in substantial reduction of narrow-sense heritability. However, these models considerably improve prediction accuracies. This was particularly significant for growth and solid-wood properties, which showed to have the highest population genetic differentiation (QST) among the studied traits. Additionally, although the pattern of correlations remained similar across the models, their magnitude was slightly lower for models that included population structure as a fixed effect. This suggests that selection, consistently performed within populations, might be less affected by unfavourable genetic correlations compared to mass selection conducted without pedigree restrictions. CONCLUSION:We conclude that the results of models properly accounting for population structure are more accurate and less biased compared to those neglecting this effect. This might have practical implications for breeders and forest managers where, decisions based on imprecise selections can pose a high risk to economic efficiency.

Wood of broad-leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. x tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis. Fungal xylanases expressed in aspen decreased lignin content in the wood of transgenic lines due to downregulation of a subset of lignin biosynthetic genes. This greatly increased saccharification potential. Transgenic plants exhibited altered growth and several signaling and regulatory genes were found affected that could mediate these effects via xylobiose signaling.

SummaryWood of broad‐leaf tree species is a valued source of renewable biomass for biorefinery and a target for genetic improvement efforts to reduce its recalcitrance. Glucuronoxylan (GX) plays a key role in recalcitrance through its interactions with cellulose and lignin. To reduce recalcitrance, we modified wood GX by expressing GH10 and GH11 endoxylanases from Aspergillus nidulans in hybrid aspen (Populus tremula L. × tremuloides Michx.) and targeting the enzymes to cell wall. The xylanases reduced tree height, modified cambial activity by increasing phloem and reducing xylem production, and reduced secondary wall deposition. Xylan molecular weight was decreased, and the spacing between acetyl and MeGlcA side chains was reduced in transgenic lines. The transgenic trees produced hypolignified xylem having thin secondary walls and deformed vessels. Glucose yields of enzymatic saccharification without pretreatment almost doubled indicating decreased recalcitrance. The transcriptomics, hormonomics and metabolomics data provided evidence for activation of cytokinin and ethylene signalling pathways, decrease in ABA levels, transcriptional suppression of lignification and a subset of secondary wall biosynthetic program, including xylan glucuronidation and acetylation machinery. Several candidate genes for perception of impairment in xylan integrity were detected. These candidates could provide a new target for uncoupling negative growth effects from reduced recalcitrance. In conclusion, our study supports the hypothesis that xylan modification generates intrinsic signals and evokes novel pathways regulating tree growth and secondary wall biosynthesis.

From an economic and environmental perspective, the use of less chemicals in the production of cellulose nanofibrils (CNFs) is advantageous. In this study, we investigated the oxidation (TEMPO/NaClO2/NaClO, pH 6.8) of softwood (SW) particles with varying amounts of TEMPO (16, 8 or 0 mg g-1 of wood). Following, TEMPO-oxidized SW nanofibrils (TO-SWNFs) were obtained by nanofibrillation and their size, morphology, and crystallite size were assessed. Hydrogel networks of TO-SWNFs were prepared and mechanical properties were measured in dH2O and phosphate buffered saline (PBS) to compare their performance for possible biomedical applications such as wound dressings. The results reveal that the presence of TEMPO is of importance for TO-SWNF network properties, presenting higher eq. H2O absorption (≈2500 %) and elongation at break (≈10 %) with good wet strength (≈180 kPa). In addition, a decrease in use of TEMPO catalyst from 16 to 8 mg g-1 of wood is possible, without detrimental effects on hydrogel network properties (dH2O absorption ≈ 2000 %, elongation at break ≈ 13 %, wet strength ≈ 190 kPa) related to applications as wound dressings.

Drought is a major factor affecting crops, thus efforts are needed to increase plant resilience to this abiotic stress. The overlapping signaling pathways between drought and cell wall integrity maintenance responses create a possibility of increasing drought resistance by modifying cell walls. Here, using herbaceous and woody plant model species, Arabidopsis and hybrid aspen, respectively, we investigated how the integrity of xylan in secondary walls affects the responses of plants to drought stress. Plants, in which secondary wall xylan integrity was reduced by expressing fungal GH10 and GH11 xylanases or by affecting genes involved in xylan backbone biosynthesis, were subjected to controlled drought while their physiological responses were continuously monitored by RGB, fluorescence, and/or hyperspectral cameras. For Arabidopsis, this was supplemented with survival test after complete water withdrawal and analyses of stomatal function and stem conductivity. All Arabidopsis xylan-impaired lines showed better survival upon complete watering withdrawal, increased stomatal density and delayed growth inhibition by moderate drought, indicating increased resilience to moderate drought associated with modified xylan integrity. Subtle differences were recorded between xylan biosynthesis mutants (irx9, irx10 and irx14) and xylanase-expressing lines. irx14 was the most drought resistant genotype, and the only genotype with increased lignin content and unaltered xylem conductivity despite its irx phenotype. Rosette growth was more affected by drought in GH11- than in GH10-expressing plants. In aspen, mild downregulation of GT43B and C genes did not affect drought responses and the transgenic plants grew better than the wild-type in drought and well-watered conditions. Both GH10 and GH11 xylanases strongly inhibited stem elongation and root growth in well-watered conditions but growth was less inhibited by drought in GH11-expressing plants than in wild-type. Overall, plants with xylan integrity impairment in secondary walls were less affected than wild-type by moderately reduced water availability but their responses also varied among genotypes and species. Thus, modifying the secondary cell wall integrity can be considered as a potential strategy for developing crops better suited to withstand water scarcity, but more research is needed to address the underlying molecular causes of this variability.

The surface temperature of a drying wooden board is strongly related to the drying process. However, it is relatively difficult to determine the surface temperature accurately during drying. In this paper, an experimental setup for analyzing the wood surface during drying by thermal imaging as well as dry and wet-bulb temperature reference surfaces was tested. Spruce sapwood samples were dried in various climates and evaluated with respect to both mass loss and surface temperature. The experimental setup enabled both qualitative and quantitative analysis of the wood drying process. The results showed that thermal imaging enabled a detailed view of the drying progression. The distinct correlation between surface temperature and mass change showed that an accurate determination of a basic, often considered, and difficult-to-determine drying potential is possible.

BACKGROUND:Wood represents the majority of the biomass on land and constitutes a renewable source of biofuels and other bioproducts. However, wood is recalcitrant to bioconversion, raising a need for feedstock improvement in production of, for instance, biofuels. We investigated the properties of wood that affect bioconversion, as well as the underlying genetics, to help identify superior tree feedstocks for biorefining. RESULTS:We recorded 65 wood-related and growth traits in a population of 113 natural aspen genotypes from Sweden ( https://doi.org/10.5061/dryad.gtht76hrd ). These traits included three growth and field performance traits, 20 traits for wood chemical composition, 17 traits for wood anatomy and structure, and 25 wood saccharification traits as indicators of bioconversion potential. Glucose release after saccharification with acidic pretreatment correlated positively with tree stem height and diameter and the carbohydrate content of the wood, and negatively with the content of lignin and the hemicellulose sugar units. Most of these traits displayed extensive natural variation within the aspen population and high broad-sense heritability, supporting their potential in genetic improvement of feedstocks towards improved bioconversion. Finally, a genome-wide association study (GWAS) revealed 13 genetic loci for saccharification yield (on a whole-tree-biomass basis), with six of them intersecting with associations for either height or stem diameter of the trees. CONCLUSIONS:The simple growth traits of stem height and diameter were identified as good predictors of wood saccharification yield in aspen trees. GWAS elucidated the underlying genetics, revealing putative genetic markers for bioconversion of bioenergy tree feedstocks.

Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large-scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five-year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2-oxoglutarate-dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4-HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP-ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild-type. The pilot-scale industrial-like pretreatment of best-performing 2OGD construct confirmed its superior sugar yields, supporting our strategy.

In the present work, it was demonstrated that mass transfer and mass transfer coefficients related to the wood drying process can be satisfactorily quantified using thermography. The method was based on continuous measurements of the wood's surface temperature, which were converted to a vapor pressure at the wood surface. The results showed that the values of the experimentally obtained transfer coefficients were in the same order of magnitude as values obtained with classical empirical correlations that apply in boundary layer theory. The measurements also showed that an average value of the mass transfer coefficient obtained during drying satisfactorily describes the complete process. The measurement set-up makes it possible to determine a surface potential accurately and continuously, which is useful in the assessment of wood drying processes.

Xylan that comprises roughly 25% of hardwood biomass is undesirable in biorefinery applications involving saccharification and fermentation. Efforts to reduce xylan levels have therefore been made in many species, usually resulting in improved saccharification. However, such modified plants have not yet been tested under field conditions. Here we evaluate the field performance of transgenic hybrid aspen lines with reduced xylan levels and assess their usefulness as short-rotation feedstocks for biorefineries. Three types of transgenic lines were tested in four-year field tests with RNAi constructs targeting either Populus GT43 clades B and C (GT43BC) corresponding to Arabidopsis clades IRX9 and IRX14, respectively, involved in xylan backbone biosynthesis, GATL1.1 corresponding to AtGALT1 involved in xylan reducing end sequence biosynthesis, or ASPR1 encoding an atypical aspartate protease. Their productivity, wood quality traits, and saccharification efficiency were analyzed. The only lines differing significantly from the wild type with respect to growth and biotic stress resistance were the ASPR1 lines, whose stems were roughly 10% shorter and narrower and leaves showed increased arthropod damage. GT43BC lines exhibited no growth advantage in the field despite their superior growth in greenhouse experiments. Wood from the ASPR1 and GT43BC lines had slightly reduced density due to thinner cell walls and, in the case of ASPR1, larger cell diameters. The xylan was less extractable by alkali but more hydrolysable by acid, had increased glucuronosylation, and its content was reduced in all three types of transgenic lines. The hemicellulose size distribution in the GALT1.1 and ASPR1 lines was skewed towards higher molecular mass compared to the wild type. These results provide experimental evidence that GATL1.1 functions in xylan biosynthesis and suggest that ASPR1 may regulate this process. In saccharification without pretreatment, lines of all three constructs provided 8-11% higher average glucose yields than wild-type plants. In saccharification with acid pretreatment, the GT43BC construct provided a 10% yield increase on average. The best transgenic lines of each construct are thus predicted to modestly outperform the wild type in terms of glucose yields per hectare. The field evaluation of transgenic xylan-reduced aspen represents an important step towards more productive feedstocks for biorefineries.

Stem bending in trees induces flexure wood but its properties and development are poorly understood. Here, we investigated the effects of low-intensity multidirectional stem flexing on growth and wood properties of hybrid aspen, and on its transcriptomic and hormonal responses. Glasshouse-grown trees were either kept stationary or subjected to several daily shakes for 5 wk, after which the transcriptomes and hormones were analyzed in the cambial region and developing wood tissues, and the wood properties were analyzed by physical, chemical and microscopy techniques. Shaking increased primary and secondary growth and altered wood differentiation by stimulating gelatinous-fiber formation, reducing secondary wall thickness, changing matrix polysaccharides and increasing cellulose, G- and H-lignin contents, cell wall porosity and saccharification yields. Wood-forming tissues exhibited elevated jasmonate, polyamine, ethylene and brassinosteroids and reduced abscisic acid and gibberellin signaling. Transcriptional responses resembled those during tension wood formation but not opposite wood formation and revealed several thigmomorphogenesis-related genes as well as novel gene networks including FLA and XTH genes encoding plasma membrane-bound proteins. Low-intensity stem flexing stimulates growth and induces wood having improved biorefinery properties through molecular and hormonal pathways similar to thigmomorphogenesis in herbaceous plants and largely overlapping with the tension wood program of hardwoods.

Important structures and functions within living organisms rely on naturally fluorescent polymeric molecules such as collagen, keratin, elastin, resilin, or lignin. Theoretical physics predict that fluorescence lifetime of these polymers is related to their chemical composition. We verified this prediction for lignin, a major structural element in plant cell walls and one of the most abundant components of wood. Lignin is composed of different types of phenylpropanoid units, and its composition affects its properties, biological functions, and the utilization of wood biomass. We carried out fluorescence lifetime imaging microscopy (FLIM) measurements of wood cell wall lignin in a population of 90 hybrid aspen trees genetically engineered to display differences in cell wall chemistry and structure. We also measured wood cell wall composition by classical analytical methods in the wood cell walls of these trees. Using statistical modelling and machine learning algorithms, we identified parameters of fluorescence lifetime that predict the content of S-type and G-type lignin units, the two main types of units in the lignin of angiosperm plants. Finally, we show how quantitative measurements of lignin chemical composition by FLIM can reveal the dynamics of lignin biosynthesis in two different biological contexts, including in vivo while lignin is being synthesized in the walls of living cells.

Wood decay is addressed continuously within thewood industry through use and development of wood preservatives.The increasing awareness on the negative effects of many chemicalstowards the environm ...