Lignification is a central process affecting plant growth, development, and stress responses. An intricate network of hormonal signals tightly regulates these processes. Several phytohormones have been demonstrated to function as important modulators of the lignin biosynthesis pathway, but their crosstalk is largely elusive. Increasing evidence of hierarchical transcriptional networks in lignification and the interaction of transcriptional regulation with different phytohormones highlights the need to emphasize this critical regulatory aspect of lignification. The hormones adjust lignification by modifying gene expression, enzyme activity, and the interaction between different metabolic pathways. This review summarizes the status of the principal hormonal control of lignification at the individual level, and also accentuates their crosstalk and the transcriptional control. Additionally, the role of reactive oxygen species (ROS) in connecting lignification with hormonal regulation is discussed. This review not only contributes to basic plant biology but also paves the way for the development of crops with improved stress tolerance and specialized lignin qualities for industrial applications.

Selection harvesting in fertile, drained peatlands is an alternative for even-aged forestry, where clearcutting takes place at the end of each rotation period. Avoiding clear-cuts has been promoted due to reducing negative externalities, like nutrient loading to waterways and significant greenhouse gas emissions after harvesting. Our aim was to understand the responses of suppressed Norway spruce trees to selection harvesting. We analysed biomass accumulation and priorities of carbon allocation to stems, proximal roots and distal roots. We quantified carbon isotope composition (delta 13C) and cross-sectional growth of proximal (supporting) roots and distal (water and nutrient transportation) roots. The study took place in a drained, fertile boreal peatland site in southern Finland, where a selection harvest was conducted in 2016, while an area as a control was left untouched. Our results show that suppressed Norway spruce trees increased their cross-sectional growth of proximal roots and distal roots twice as much as that of the stem during the first five years after the selection harvest. The timing and magnitude of carbon allocation to proximal roots and distal roots were immediate and equal, underlining the fact that trees were investing in mechanical support and nutrient and water uptake. These results show that the climate benefits of selection harvest were not delayed. Instead of immediate growth of stems, we found immediate growth of roots.

Investigations to uncover the versatility of carbohydrate active enzymes belonging to the auxiliary activity family 3 (AA3) have focused on microbial enzymes. Plant genomes also harbor AA3 encoding genes, and reverse genetics approaches have shown their importance in plant development. To date, however, detailed biochemical characterizations and structures of plant AA3 enzymes have not been reported. Here, we describe screening AA3 encoding genes from Norway spruce and the first in-depth biochemical and structural characterization of a plant AA3 oxidase, PaAOX1. PaAOX1 was crystallized, and the structure was solved by X-ray crystallography. PaAOX1 demonstrated highest oxidase activity against glycerol. It oxidized glycerol to glyceraldehyde, then further to glyceric acid, and showed preference for L-glyceraldehyde over D-glyceraldehyde. Despite the low sequence similarity with fungal alcohol oxidases, it had all the structural features of the typical AA3 enzyme. PaAOX1 has a novel activity among the characterized AA3s, and it showed a new structural arrangement of the region surrounding the catalytic center. Our findings contribute to a deeper understanding of the structural and functional aspects of enzymes in the GMC superfamily, and especially of alcohol oxidases, expanding the knowledge from fungi to plants. ### Competing Interest Statement The authors have declared no competing interest.

Nanoparticles from biobased raw materials can be utilised to improve functional properties of textiles and fibrebased packaging materials. Safety of chemicals need to be assessed in the EU before entering to the market. This study investigates the bioactivity and potential ecotoxicity and toxicity of biobased nanoparticles from hydrolyzed suberin (SNPs), softwood kraft lignin (lignin nanoparticles, LNPs), and tall oil fatty acid esterified softwood kraft lignin (TOFA-lignin, TOFA-LNPs). Nanoparticles were assayed as concentrations sufficient for textile coating applications for cytotoxicity, skin sensitization, corrosion, and irritation in vitro. For ecotoxicity evaluation, algal inhibition, Daphnia sp. acute immobilization, D. magna reproduction, and toxicity to earthworms were tested. Antibacterial properties were examined using recombinant Escherichia coli and Staphylococcus aureus strains, while antioxidant activities were evaluated with in vitro assays. Results indicated that nanoparticles were safe at the studied concentrations according to the cytotoxicity and skin irritation or corrosion tests. However, LNPs at concentrations of 70.31 mu g/mL and TOFA-LNPs at 125 mu g/mL (w/v) showed skin sensitization, unlike SNPs. Ecotoxicity assays revealed that all aqueous nanoparticle dispersions exhibited effects on algae and daphnids but were harmless to earthworms at the concentrations tested. LNPs and TOFA-LNPs showed high antibacterial and antioxidant activities, which can likely be attributed to their high total phenolic content. With structurally more complex SNPs, both antioxidant and antibacterial activities were low. The results show that the investigated nanoparticles are potential alternatives to fossil-based and more harmful chemicals. Biobased, safeassessed alternatives for coating of textiles could be a way to increase the environmentally sustainability.

Using microarray polymer profiling (MAPP), immunocytochemical staining, and Raman spectroscopy, we conducted a detailed spatiotemporal survey of the deposition of cell wall components during xylem differentiation in Norway spruce. These insights are essential for understanding the intricate architecture of wood cell walls during the initial stages of development immediately following cambial divisions. MAPP was used to obtain an overview of cell wall polysaccharides, glycoproteins, and ferulic acid residues in developing xylem. The most interesting findings were investigated in further detail using immunomicroscopy and Raman spectroscopy. In axial tracheids, deposition of pectic compounds and cellulose only slightly preceded the first deposition of lignin. Lignification was initiated in the middle lamellae and cell wall corners and proceeded during the development of the S1 layer and intensified later during cell wall thickening. Raman analysis revealed an earlier deposition of lignin than shown previously, which coincided with the deposition of methylesterified or partially methylesterified homogalacturonan initially in the radial cell walls and later in the tangential cell walls of developing earlywood. Minimal binding of an antibody against feruloylated compounds indicated that ferulic acid residues do not participate in the initiation of lignification in Norway spruce.

Bark is one of the key sidestreams produced in sawmills and currently is typically used for energy generation. Storage of bark is required to ensure supply at times of high heating demand, i.e. in cold winter months. In addition to energy generation, mono-, sesqui-, and diterpenes are components of resin in coniferous trees that can be used in various applications. The main aim of the present work was to investigate emissions of mono- and sesquiterpenes and common greenhouse gases, their generation, and flux dynamics within stockpiles of Scots pine (Pinus sylvestris L.) bark occurring during long-term storage in outdoor conditions. The focus was therefore on conversions of organic matter by decomposition into various gaseous substances. The work adds to the understanding how the decomposition products and volatile organic compounds (VOCs) are emitted from a stockpile and their stockpile pore space gas dynamics. Furthermore, it demonstrates how ventilation of a stockpile can affect the dynamics of decomposition product release and timing of VOC emissions during the storage. The storage time lasted from mid-April until the end of September 2021, during which seven samplings were carried out. CO2 was the main greenhouse gas liberated, with some emission of CH4, CO, and methanol. The major content of monoterpenes was released in the first 11 days after the stockpile establishment, and approximately half the sesquiterpenes in a 50-day storage time. Ventilation showed an effect on the release in the analysed cases, as the compounds decreased more rapidly over time due to ventilation. While the main share of carbon (C) losses originated from the release through CO2 in the non-ventilated stockpile, approximately 39 % more C was released in the ventilated stockpile. Gases other than CO2 were responsible for about 1 % of the total gaseous C losses from the non-ventilated, and 2 % from the ventilated, stockpile.

Lignin is a phenolic polymer in plants that rigidifies the cell walls of water-conducting tracheary elements and support-providing fibers and stone cells. Different mechanisms have been suggested for the transport of lignin precursors to the site of lignification in the cell wall. Extracellular vesicle (EV)-enriched samples isolated from a lignin-forming cell suspension culture of Norway spruce (Picea abies L. Karst.) contained both phenolic metabolites and enzymes related to lignin biosynthesis. Metabolomic analysis revealed mono-, di-, and oligolignols in the EV isolates, as well as carbohydrates and amino acids. In addition, salicylic acid (SA) and some proteins involved in SA signaling were detected in the EV-enriched samples. A proteomic analysis detected several laccases, peroxidases, beta-glucosidases, putative dirigent proteins, and cell wall-modifying enzymes, such as glycosyl hydrolases, transglucosylase/hydrolases, and expansins in EVs. Our findings suggest that EVs are involved in transporting enzymes required for lignin polymerization in Norway spruce, and radical coupling of monolignols can occur in these vesicles. Extracellular vesicles transport enzymes putatively involved in lignin polymerization in Norway spruce, and radical coupling of monolignols can occur in these vesicles.

In order to develop more economic uses of lignin, greater knowledge regarding its native structure is required. This can inform the development of optimized extraction methods that preserve desired structural properties. Current extraction methods alter the polymeric structure of lignin, leading to a loss of valuable structural groups or the formation of new non-native ones. In this study, Norway spruce (Picea abies) tissue-cultured cells that produce lignin extracellularly in a suspension medium were employed. This system enables the investigation of unaltered native lignin, as no physicochemical extraction steps are required. For the first time, this culture was used to investigate the interactions between lignin and xylan, a secondary cell wall hemicellulose, and to study the importance of lignin-carbohydrate complexes (LCCs) on the polymerization and final structure of extracellular lignin (ECL). This has enabled us to study the impact of xylan on monolignol composition and structure of the final lignin polymer. We find that the addition of xylan to the solid culture medium accelerates cell growth and impacts the ratio of monolignols in the lignin. However, the presence of xylan in the lignin polymerization environment does not significantly alter the structural properties of lignin as analyzed by two-dimensional nuclear magnetic resonance (NMR) spectroscopy and size exclusion chromatography (SEC). Nevertheless, our data indicate that xylan can act as a nucleation point, leading to more rapid lignin polymerization, an important insight into biopolymer interactions during cell wall synthesis in wood. Lignin structure and interactions with a secondary cell wall hemicellulose were investigated in a model cell culture: we found that the polymerization and final structure of lignin are altered when the hemicellulose is present during cell growth and monolignol production. The physicochemical interactions between lignin and xylan partly define the extractability and utility of native lignin in high value applications, so this work has implications for lignin extraction as well as fundamental plant biology.

The blackening of cut carrots causes substantial economic losses to the food industry. Blackening was not observed in carrots that had been stored underground for less than a year, but the susceptibility to blackening increased with the age of the carrots that were stored underground for longer periods. Samples of black, border, and orange tissues from processed carrot batons and slices, prepared under industry standard conditions, were analyzed to identify the molecular and metabolic mechanisms underpinning processing-induced blackening. The black tissues showed substantial molecular and metabolic rewiring and large changes in the cell wall structure, with a decreased abundance of xyloglucan, pectins (homogalacturonan, rhamnogalacturonan-I, galactan and arabinan), and higher levels of lignin and other phenolic compounds when compared to orange tissues. Metabolite profiling analysis showed that there was a major shift from primary to secondary metabolism in the black tissues, which were depleted in sugars, amino acids, and tricarboxylic acid (TCA) cycle intermediates but were rich in phenolic compounds. These findings suggest that processing triggers a release from quiescence. Transcripts encoding proteins associated with secondary metabolism were less abundant in the black tissues, but there were no increases in transcripts associated with oxidative stress responses, programmed cell death, or senescence. We conclude that restraining quiescence release alters cell wall metabolism and composition, particularly regarding pectin composition, in a manner that increases susceptibility to blackening upon processing.

Plant respiratory burst oxidase homologs (RBOHs) are plasma membrane-localized NADPH oxidases that generate superoxide anion radicals, which then dismutate to H2O2, into the apoplast using cytoplasmic NADPH as an electron donor. PaRBOH1 is the most highly expressed RBOH gene in developing xylem as well as in a lignin-forming cell culture of Norway spruce (Picea abies L. Karst.). Since no previous information about regulation of gymnosperm RBOHs exist, our aim was to resolve how PaRBOH1 is regulated with a focus on phosphorylation. The N-terminal part of PaRBOH1 was found to contain several putative phosphorylation sites and a four-times repeated motif with similarities to the Botrytis-induced kinase 1 target site in Arabidopsis AtRBOHD. Phosphorylation was indicated for six of the sites in in vitro kinase assays using 15 amino-acid-long peptides for each of the predicted phosphotarget site in the presence of protein extracts of developing xylem. Serine and threonine residues showing positive response in the peptide assays were individually mutated to alanine (kinase-inactive) or to aspartate (phosphomimic), and the wild type PaRBOH1 and the mutated constructs transfected to human kidney embryogenic (HEK293T) cells with a low endogenous level of extracellular ROS production. ROS-producing assays with HEK cells showed that Ca2+ and phosphorylation synergistically activate the enzyme and identified several serine and threonine residues that are likely to be phosphorylated including a novel phosphorylation site not characterized in other plant species. These were further investigated with a phosphoproteomic study. Results of Norway spruce, the first gymnosperm species studied in relation to RBOH regulation, show that regulation of RBOH activity is conserved among seed plants.

The fiber length, chemical composition, and mycototoxins of oat and barley hulls were analyzed in order to study the suitability to utilize hulls for paper and paperboard manufacturing. Oat fibers were observed to be slightly longer than those of barley (0.80 ± 0.19 mm and 0.51 ± 0.17 mm, respectively). There were no differences in the composition of the hemicellulose sugars, with the predominance of xylose and glucose. The amount of Klason lignin in oat and barley hulls was at almost the same level (23% and 25% of dry weight, respectively). Guaiacyl (G) units derived from coniferyl alcohol dominated as shown by pyrolysis-GC-MS. To achieve fibrillation beneficial for the papermaking, oat and barley hulls were refined with a disc refiner. Oat hulls were observed to be harder than barley hulls. This affected both calendering and the appearance of the sheets produced. Laboratory sheets were prepared in a sheet-former with different percentage of softwood pulp and oat and barley hulls. Hull-containing paper mostly met the same quality requirements as the commercial paper except for the burst strength in sheets containing either species’ hulls, and the tensile strength in sheets containing barley hulls.

Lignans are bioactive compounds that are especially abundant in the Norway spruce (Picea abiesL. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and imaging techniques, we were able to quantify, qualify and localise the easily extractable lignans in the xylem tissue. The knotwood samples contained 15 different lignans according to the gas chromatography-mass spectrometry analysis. They comprised 16% of the knotwood dry weight and 82% of the acetone extract. The main lignans were found to be hydroxymatairesinols HMR1 and HMR2. Cryosectioned and resin-embedded ultrathin sections of the knotwood were analysed with scanning transmission X-ray microscopy (STXM). Cryosectioning was found to retain only lignan residues inside the cell lumina. In the resin-embedded samples, lignan was interpreted to be unevenly distributed inside the cell lumina, and partially confined in deposits which were either readily present in the lumina or formed when OsO(4)used in staining reacted with the lignans. Furthermore, the multi-technique characterisation enabled us to obtain information on the chemical composition of the structural components of knotwood. A simple spectral analysis of the STXM data gave consistent results with the gas chromatographic methods about the relative amounts of cell wall components (lignin and polysaccharides). The STXM analysis also indicated that a torus of a bordered pit contained aromatic compounds, possibly lignin.

Finnish N fertilizer application regulations for forage grasses are based on field experiments mainly conducted in the 1960-1970s with cultivars and management practices typical of the time. In order to update the yield response function of N, to make it better suited to current grassland farming, field experiments were conducted at two sites in 2015-2017 with two cultivars of timothy (Phleum pratense L.) and one of meadow fescue (Festuca pratensis Huds.). Dry matter (DM) yield, nutritive value and N balance were evaluated, with N application levels 0, 150, 200, 250, 300, 350, 400 and 450 kg N ha(-1) year(-1). The grasses were harvested three times per season. The data indicate that the DM yield response was significantly stronger, and N was used more efficiently for DM production than earlier without compromising the nutritive value, especially during the first two years. The third harvest produced on average 23% of the annual yield, utilizing N efficiently. N application rates below 350 kg N ha(-1) year(-1) did not cause substantial overwintering losses or lodging. The data indicate that with changing climate and improved cultivars and management practices, there is a need to modify the rates and timing of N application. The results suggest that N application levels could be increased by at least 50 kg N ha(-1) year(-1) from the current maximum accepted rate (250 kg N ha(-1) year(-1)) without too high NO3- or CP concentrations in feed, or too high N balance that indicates increasing risk of N leaching.

Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissue-cultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tonoplast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the Km value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters.

In the version of this article initially published, there was a mistake in the calculation of the nucleotide mutation rate per site per generation: 1 × 10 −9 mutations per site per generation was used, whereas 9.5 × 10 −9 was correct. This error affects the interpretation of population-size changes over time and their possible correspondence with known geological events, as shown in the original Fig. 4 and supporting discussion in the text, as well as details in the Supplementary Note. Neither the data themselves nor any other results are affected. Figure 4 has been revised accordingly. Images of the original and corrected figure panels are shown in the correction notice.

Direct evidence of lignin–carbohydrate linkages was found in lignin substrates isolated and synthetized mimicking their native state and overcoming any kind of harsh extraction and chemical pre-treatments.

A comparative transcriptomic study and a single-cell metabolome analysis were combined to determine whether parenchymal ray cells contribute to the biosynthesis of monolignols in the lignifying xylem of Norway spruce (Picea abies). Ray parenchymal cells may function in the lignification of upright tracheids by supplying monolignols. To test this hypothesis, parenchymal ray cells and upright tracheids were dissected with laser-capture microdissection from tangential cryosections of developing xylem of spruce trees. The transcriptome analysis revealed that among the genes involved in processes typical for vascular tissues, genes encoding cell wall biogenesis-related enzymes were highly expressed in both developing tracheids and ray cells. Interestingly, most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Nonetheless, 1,073 differentially expressed genes were detected between developing ray cells and tracheids, among which a set of genes expressed only in ray cells was identified. In situ single cell metabolomics of semi-intact plants by picoliter pressure probe-electrospray ionization-mass spectrometry detected monolignols and their glycoconjugates in both cell types, indicating that the biosynthetic route for monolignols is active in both upright tracheids and parenchymal ray cells. The data strongly support the hypothesis that in developing xylem, ray cells produce monolignols that contribute to lignification of tracheid cell walls.

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