Different combinations of laccase paralogs nonredundantly control the amount and composition of lignin in specific cell types and cell wall layers in Arabidopsis.

Vascular plants reinforce the cell walls of the different xylem cell types with lignin phenolic polymers. Distinct lignin chemistries differ between each cell wall layer and each cell type to support their specific functions. Yet the mechanisms controlling the tight spatial localization of specific lignin chemistries remain unclear. Current hypotheses focus on control by monomer biosynthesis and/or export, while cell wall polymerization is viewed as random and nonlimiting. Here, we show that combinations of multiple individual laccases (LACs) are nonredundantly and specifically required to set the lignin chemistry in different cell types and their distinct cell wall layers. We dissected the roles of Arabidopsis thaliana LAC4, 5, 10, 12, and 17 by generating quadruple and quintuple loss-of-function mutants. Loss of these LACs in different combinations led to specific changes in lignin chemistry affecting both residue ring structures and/or aliphatic tails in specific cell types and cell wall layers. Moreover, we showed that LAC-mediated lignification has distinct functions in specific cell types, waterproofing fibers, and strengthening vessels. Altogether, we propose that the spatial control of lignin chemistry depends on different combinations of LACs with nonredundant activities immobilized in specific cell types and cell wall layers.

Monolignol export by diffusion down a polymerization-induced concentration gradient.

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. A key element of lignin biosynthesis is unknown: how do lignin precursors (monolignols) get from inside the cell out to the cell wall where they are polymerized? Modeling indicates that monolignols can passively diffuse through lipid bilayers, but this has not been tested experimentally. We demonstrate significant monolignol diffusion occurs when laccases, which consume monolignols, are present on one side of the membrane. We hypothesize that lignin polymerization could deplete monomers in the wall, creating a concentration gradient driving monolignol diffusion. We developed a two-photon microscopy approach to visualize lignifying Arabidopsis thaliana root cells. Laccase mutants with reduced ability to form lignin polymer in the wall accumulated monolignols inside cells. In contrast, active transport inhibitors did not decrease lignin in the wall and scant intracellular phenolics were observed. Synthetic liposomes were engineered to encapsulate laccases, and monolignols crossed these pure lipid bilayers to form polymer within. A sink-driven diffusion mechanism explains why it has been difficult to identify genes encoding monolignol transporters and why the export of varied phenylpropanoids occurs without specificity. It also highlights an important role for cell wall oxidative enzymes in monolignol export.

The Output of Protein-Coding Genes Shifts to Circular RNAs When the Pre-mRNA Processing Machinery Is Limiting.

Many eukaryotic genes generate linear mRNAs and circular RNAs, but it is largely unknown how the ratio of linear to circular RNA is controlled or modulated. Using RNAi screening in Drosophila cells, we identify many core spliceosome and transcription termination factors that control the RNA outputs of reporter and endogenous genes. When spliceosome components were depleted or inhibited pharmacologically, the steady-state levels of circular RNAs increased while expression of their associated linear mRNAs concomitantly decreased. Upon inhibiting RNA polymerase II termination via depletion of the cleavage/polyadenylation machinery, circular RNA levels were similarly increased. This is because readthrough transcripts now extend into downstream genes and are subjected to backsplicing. In total, these results demonstrate that inhibition or slowing of canonical pre-mRNA processing events shifts the steady-state output of protein-coding genes toward circular RNAs. This is in part because nascent RNAs become directed into alternative pathways that lead to circular RNA production.

Explosive seed dispersal depends on SPL7 to ensure sufficient copper for localized lignin deposition via laccases.

Exploding seed pods evolved in the Arabidopsis relative Cardamine hirsuta via morphomechanical innovations that allow the storage and rapid release of elastic energy. Asymmetric lignin deposition within endocarpb cell walls is one such innovation that is required for explosive seed dispersal and evolved in association with the trait. However, the genetic control of this novel lignin pattern is unknown. Here, we identify three lignin-polymerizing laccases, LAC4, 11, and 17, that precisely colocalize with, and are redundantly required for, asymmetric lignification of endocarpb cells. By screening for C. hirsuta mutants with less lignified fruit valves, we found that loss of function of the transcription factor gene SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 7 (SPL7) caused a reduction in endocarpb cell-wall lignification and a consequent reduction in seed dispersal range. SPL7 is a conserved regulator of copper homeostasis and is both necessary and sufficient for copper to accumulate in the fruit. Laccases are copper-requiring enzymes. We discovered that laccase activity in endocarpb cell walls depends on the SPL7 pathway to acclimate to copper deficiency and provide sufficient copper for lignin polymerization. Hence, SPL7 links mineral nutrition to efficient dispersal of the next generation.

Molecular identification of a laccase that catalyzes the oxidative coupling of a hydroxycinnamic acid amide for hordatine biosynthesis in barley.

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound accumulated at high levels in young barley (Hordeum vulgare) seedlings. The enzyme responsible for the oxidative dimerization of pCA, which is the final step of the hordatine biosynthetic pathway, has not been identified. In this study, we first verified the presence of this enzyme activity in the crude extract of barley seedlings. Because the enzyme activity was not dependent on H2 O2 , the responsible enzyme was not peroxidase, which was previously implicated in HA biosynthesis. The analysis of the dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected HA in the wheat lines carrying the distal part of the 2H short arm. This chromosomal region contains two laccase genes (HvLAC1 and HvLAC2) that are highly expressed at the seedling stage and may encode enzymes that oxidize pCA during the formation of HA. Changes in the HvLAC transcript levels coincided with the changes in the HA biosynthesis-related enzyme activities in the crude extract and the HA content in barley seedlings. Moreover, HvLAC genes were heterologously expressed in Nicotiana benthamiana leaves and in bamboo (Phyllostachys nigra) suspension cells and HA biosynthetic activities were detected in the crude extract of transformed N. benthamiana leaves and bamboo suspension cells. The HA formed by the enzymatic reaction had the same stereo-configuration as the naturally occurring HA. These results demonstrate that HvLAC enzymes mediate the oxidative coupling of pCA during HA biosynthesis.

Uncovering the lignin-degrading potential of Serratia quinivorans AORB19: insights from genomic analyses and alkaline lignin degradation.

BACKGROUND: Lignin is an intricate phenolic polymer found in plant cell walls that has tremendous potential for being converted into value-added products with the possibility of significantly increasing the economics of bio-refineries. Although lignin in nature is bio-degradable, its biocatalytic conversion is challenging due to its stable complex structure and recalcitrance. In this context, an understanding of strain's genomics, enzymes, and degradation pathways can provide a solution for breaking down lignin to unlock the full potential of lignin as a dominant valuable bioresource. A gammaproteobacterial strain AORB19 has been isolated previously from decomposed wood based on its high laccase production. This work then focused on the detailed genomic and functional characterization of this strain based on whole genome sequencing, the identification of lignin degradation products, and the strain's laccase production capabilities on various agro-industrial residues. RESULTS: Lignin degrading bacterial strain AORB19 was identified as Serratia quinivorans based on whole genome sequencing and core genome phylogeny. The strain comprised a total of 123 annotated CAZyme genes, including ten cellulases, four hemicellulases, five predicted carbohydrate esterase genes, and eight lignin-degrading enzyme genes. Strain AORB19 was also found to possess genes associated with metabolic pathways such as the ß-ketoadipate, gentisate, anthranilate, homogentisic, and phenylacetate CoA pathways. LC-UV analysis demonstrated the presence of p-hydroxybenzaldehyde and vanillin in the culture media which constitutes potent biosignatures indicating the strain's capability to degrade lignin. Finally, the study evaluated the laccase production of Serratia AORB19 grown with various industrial raw materials, with the highest activity detected on flax seed meal (257.71 U/L), followed by pea hull (230.11 U/L), canola meal (209.56 U/L), okara (187.67 U/L), and barley malt sprouts (169.27 U/L). CONCLUSIONS: The whole genome analysis of Serratia quinivorans AORB19, elucidated a repertoire of genes, pathways and enzymes vital for lignin degradation that widens the understanding of ligninolytic metabolism among bacterial lignin degraders. The LC-UV analysis of the lignin degradation products coupled with the ability of S. quinivorans AORB19 to produce laccase on diverse agro-industrial residues underscores its versatility and its potential to contribute to the economic viability of bio-refineries.

Identification of a novel marker and its associated laccase gene for regulating ear length in tropical and subtropical maize lines.

KEY MESSAGE: This study revealed the identification of a novel gene, Zm00001d042906, that regulates maize ear length by modulating lignin synthesis and reported a molecular marker for selecting maize lines with elongated ears. Maize ear length has garnered considerable attention due to its high correlation with yield. In this study, six maize inbred lines of significant importance in maize breeding were used as parents. The temperate maize inbred line Ye107, characterized by a short ear, was crossed with five tropical or subtropical inbred lines featuring longer ears, creating a multi-parent population displaying significant variations in ear length. Through genome-wide association studies and mutation analysis, the A/G variation at SNP_183573532 on chromosome 3 was identified as an effective site for discriminating long-ear maize. Furthermore, the associated gene Zm00001d042906 was found to correlate with maize ear length. Zm00001d042906 was functionally annotated as a laccase (Lac4), which showed activity and influenced lignin synthesis in the midsection cells of the cob, thereby regulating maize ear length. This study further reports a novel molecular marker and a new gene that can assist maize breeding programs in selecting varieties with elongated ears.

A complex metabolic network and its biomarkers regulate laccase production in white-rot fungus Cerrena unicolor 87613.

BACKGROUND: White-rot fungi are known to naturally produce high quantities of laccase, which exhibit commendable stability and catalytic efficiency. However, their laccase production does not meet the demands for industrial-scale applications. To address this limitation, it is crucial to optimize the conditions for laccase production. However, the regulatory mechanisms underlying different conditions remain unclear. This knowledge gap hinders the cost-effective application of laccases. RESULTS: In this study, we utilized transcriptomic and metabolomic data to investigate a promising laccase producer, Cerrena unicolor 87613, cultivated with fructose as the carbon source. Our comprehensive analysis of differentially expressed genes (DEGs) and differentially abundant metabolites (DAMs) aimed to identify changes in cellular processes that could affect laccase production. As a result, we discovered a complex metabolic network primarily involving carbon metabolism and amino acid metabolism, which exhibited contrasting changes between transcription and metabolic patterns. Within this network, we identified five biomarkers, including succinate, serine, methionine, glutamate and reduced glutathione, that played crucial roles in co-determining laccase production levels. CONCLUSIONS: Our study proposed a complex metabolic network and identified key biomarkers that determine the production level of laccase in the commercially promising Cerrena unicolor 87613. These findings not only shed light on the regulatory mechanisms of carbon sources in laccase production, but also provide a theoretical foundation for enhancing laccase production through strategic reprogramming of metabolic pathways, especially related to the citrate cycle and specific amino acid metabolism.

Genome-wide screen identifies new set of genes for improved heterologous laccase expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is widely used as a host cell for recombinant protein production due to its fast growth, cost-effective culturing, and ability to secrete large and complex proteins. However, one major drawback is the relatively low yield of produced proteins compared to other host systems. To address this issue, we developed an overlay assay to screen the yeast knockout collection and identify mutants that enhance recombinant protein production, specifically focusing on the secretion of the Trametes trogii fungal laccase enzyme. Gene ontology analysis of these mutants revealed an enrichment of processes including vacuolar targeting, vesicle trafficking, proteolysis, and glycolipid metabolism. We confirmed that a significant portion of these mutants also showed increased activity of the secreted laccase when grown in liquid culture. Notably, we found that the combination of deletions of OCA6, a tyrosine phosphatase gene, along with PMT1 or PMT2, two genes encoding ER membrane protein-O-mannosyltransferases involved in ER quality control, and SKI3, which encode for a component of the SKI complex responsible for mRNA degradation, further increased secreted laccase activity. Conversely, we also identified over 200 gene deletions that resulted in decreased secreted laccase activity, including many genes that encode for mitochondrial proteins and components of the ER-associated degradation pathway. Intriguingly, the deletion of the ER DNAJ co-chaperone gene SCJ1 led to almost no secreted laccase activity. When we expressed SCJ1 from a low-copy plasmid, laccase secretion was restored. However, overexpression of SCJ1 had a detrimental effect, indicating that precise dosing of key chaperone proteins is crucial for optimal recombinant protein expression. This study offers potential strategies for enhancing the overall yield of recombinant proteins and provides new avenues for further research in optimizing protein production systems.

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Detoxification of tetracycline and synthetic dyes by a newly characterized Lentinula edodes laccase, and safety assessment using proteomic analysis.

Fungal laccase has strong ability in detoxification of many environmental contaminants. A putative laccase gene, LeLac12, from Lentinula edodes was screened by secretome approach. LeLac12 was heterogeneously expressed and purified to characterize its enzymatic properties to evaluate its potential use in bioremediation. This study showed that the extracellular fungal laccase from L. edodes could effectively degrade tetracycline (TET) and the synthetic dye Acid Green 25 (AG). The growth inhibition of Escherichia coli and Bacillus subtilis by TET revealed that the antimicrobial activity was significantly reduced after treatment with the laccase-HBT system. 16 transformation products of TET were identified by UPLC-MS-TOF during the laccase-HBT oxidation process. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that LeLac12 could completely mineralize ring-cleavage products. LeLac12 completely catalyzed 50 mg/L TET within 4 h by adding AG (200 mg/L), while the degradation of AG was above 96% even in the co-contamination system. Proteomic analysis revealed that central carbon metabolism, energy metabolism, and DNA replication/repair were affected by TET treatment and the latter system could contribute to the formation of multidrug-resistant strains. The results demonstrate that LeLac12 is an efficient and environmentally method for the removal of antibiotics and dyes in the complex polluted wastewater.

Combined Strategies for Improving Aflatoxin B1 Degradation Ability and Yield of a Bacillus licheniformis CotA-Laccase.

Aflatoxin B1 (AFB1) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from Bacillus licheniformis ANSB821 previously reported by our laboratory showed great potential to degrade AFB1 without redox mediators. However, the use of this CotA-laccase to remove AFB1 in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB1, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB1, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (kcat/Km) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into Pichia pastoris GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in P. pastoris GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB1 detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.

[Monogenic variants in Laccase domain-containing 1 (LACC1) as the cause of juvenile arthritis]. / Monogene Varianten in "laccase domain-containing 1" (LACC1) als Ursache einer juvenilen Arthritis.

Monogenic mutations in laccase domain-containing 1 (LACC1) are associated with clinical pictures that mimic severe courses of polyarticular or systemic juvenile idiopathic arthritis. The diseases are characterized by an early onset during the first year of life, a familial clustering and a high inflammatory activity. The courses are mostly difficult to influence and often lead to sequelae. In this article four cases from two families are presented in which the homozygous mutation p.T276fs* in LACC1 was detected. The children initially suffered from polyarticular or systemic forms of juvenile arthritis. Of the patients two are currently being treated with tocilizumab and methotrexate and one female patient without a basis treatment is currently only receiving local repeated intra-articular steroids. A fourth female patient underwent an allogeneic bone marrow transplantation due to a relapse of an acute lymphatic leukemia. Since then, no further inflammatory symptoms have occurred. The cases presented are compared with the other 50 courses published to date. In addition, recent studies investigating the influence of LACC1 mutations, particularly on macrophage function, are summarized.

Genome-wide study of Cerrena unicolor 87613 laccase gene family and their mode prediction in association with substrate oxidation.

BACKGROUND: Laccases are green biocatalysts with wide industrial applications. The study of efficient and specific laccase producers remains a priority. Cerrena species have been shown to be promising basidiomycete candidates for laccase production. Although two sets of Cerrena genome data have been publicly published, no comprehensive bioinformatics study of laccase gene family in C. unicolor has been reported, particularly concerning the analysis of their three-dimensional (3D) structures and molecular docking to substrates, like ABTS and aflatoxin B1 (AFB1). RESULTS: In this study, we conducted a comprehensive genome-wide analysis of laccase gene family in C. unicolor 87613. We identified eighteen laccase genes (CuLacs) and classified them into three clades using phylogenetic analysis. We characterized these laccases, including their location in contig 5,6,9,12,15,19,26,27, gene structures of different exon-intron arrangements, molecular weight ranging from 47.89 to 141.41 kDa, acidic pI value, 5-15 conserved protein motifs, signaling peptide of extracellular secretion (harbored by 13 CuLacs) and others. In addition, the analysis of cis-acting element in laccase promoters indicated that the transcription response of CuLac gene family was regulatable and complex under different environmental cues. Furthermore, analysis of transcription pattern revealed that CuLac8, 12 and CuLac2, 13 were the predominant laccases in response to copper ions or oxidative stress, respectively. Finally, we focused on the 3D structure analysis of CuLac proteins. Seven laccases with extra transmembrane domains or special sequences were particularly interesting. Predicted structures of each CuLac protein with or without these extra sequences showed altered interacting amino acid residues and binding sites, leading to varied affinities to both ABTS and AFB1. As far as we know, it is the first time to discuss the influence of the extra sequence on laccase's affinity to substrates. CONCLUSIONS: Our findings provide robust genetic data for a better understanding of the laccase gene family in C. unicolor 87613, and create a foundation for the molecular redesign of CuLac proteins to enhance their industrial applications.

Comparative genomic analysis of five coprinoid mushrooms species.

Although coprinoid mushrooms are widely known for the phenomenon of deliquescence and production of fungal laccases and extracellular peroxygenases, the genome structure and genetic diversity of coprinoid mushroom species have not been extensively studied. To reveal the genomic structure and diversity in coprinoid mushroom species, the genomes of five coprinoid mushroom species were compared and analyzed. A total of 24,303 orthologous gene families, including 89,462 genes, were identified in the five species. The numbers of core, softcore, dispensable, and private genes were 5617 (25.6%), 1628 (7.4%), 2083 (9.5%), and 12,574 (57.4%), respectively. Differentiation time analysis revealed that Coprinellus micaceus and Coprinellus angulatus differentiated approximately 181.0 million years ago. Coprinopsis cinerea and Coprinopsis marcescibilis differentiated approximately 131.0 million years ago, and they were differentiated from Candolleomyces aberdarensis approximately 176.0 million years ago. Gene family contraction and expansion analyses showed that 1465 genes and 532 gene families were expanded, and 95 genes and 134 gene families were contracted. Ninety-five laccase-coding genes were detected in the five species, and the distribution of the laccase-coding genes in the five species was not uniform. These data provide a reference for a deeper understanding of the genetic structure of the genomes of coprinoid mushroom species. Furthermore, this study provides a reference for follow-up studies on the genome structure of coprinoid mushroom species and the diversity of specific functional genes.

Comprehensive analysis of the laccase gene family in tea plant highlights its roles in development and stress responses.

BACKGROUND: Laccase (LAC) is the pivotal enzyme responsible for the polymerization of monolignols and stress responses in plants. However, the roles of LAC genes in plant development and tolerance to diverse stresses are still largely unknown, especially in tea plant (Camellia sinensis), one of the most economically important crops worldwide. RESULTS: In total, 51 CsLAC genes were identified, they were unevenly distributed on different chromosomes and classified into six groups based on phylogenetic analysis. The CsLAC gene family had diverse intron-exon patterns and a highly conserved motif distribution. Cis-acting elements in the promoter demonstrated that promoter regions of CsLACs encode various elements associated with light, phytohormones, development and stresses. Collinearity analysis identified some orthologous gene pairs in C. sinensis and many paralogous gene pairs among C. sinensis, Arabidopsis and Populus. Tissue-specific expression profiles revealed that the majority of CsLACs had high expression in roots and stems and some members had specific expression patterns in other tissues, and the expression patterns of six genes by qRT‒PCR were highly consistent with the transcriptome data. Most CsLACs showed significant variation in their expression level under abiotic (cold and drought) and biotic (insect and fungus) stresses via transcriptome data. Among them, CsLAC3 was localized in the plasma membrane and its expression level increased significantly at 13 d under gray blight treatment. We found that 12 CsLACs were predicted to be targets of cs-miR397a, and most CsLACs showed opposite expression patterns compared to cs-miR397a under gray blight infection. Additionally, 18 highly polymorphic SSR markers were developed, these markers can be widely used for diverse genetic studies of tea plants. CONCLUSIONS: This study provides a comprehensive understanding of the classification, evolution, structure, tissue-specific profiles, and (a)biotic stress responses of CsLAC genes. It also provides valuable genetic resources for functional characterization towards enhancing tea plant tolerance to multiple (a)biotic stresses.

Genome-wide identification and expression patterns of the laccase gene family in response to kiwifruit bacterial canker infection.

BACKGROUND: Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a destructive disease worldwide. Resistance genes that respond to Psa infection urgently need to be identified for controlling this disease. Laccase is mainly involved in the synthesis of lignin in the plant cell wall and plays a prominent role in plant growth and resistance to pathogen infection. However, the role of laccase in kiwifruit has not been reported, and whether laccase is pivotal in the response to Psa infection remains unclear. RESULTS: We conducted a bioinformatics analysis to identify 55 laccase genes (AcLAC1-AcLAC55) in the kiwifruit genome. These genes were classified into five cluster groups (I-V) based on phylogenetic analysis, with cluster groups I and II having the highest number of members. Analysis of the exon-intron structure revealed that the number of exons varied from 1 to 8, with an average of 5 introns. Our evolutionary analysis indicated that fragment duplication played a key role in the expansion of kiwifruit laccase genes. Furthermore, evolutionary pressure analysis suggested that AcLAC genes were under purifying selection. We also performed a cis-acting element analysis and found that AcLAC genes contained multiple hormone (337) and stress signal (36) elements in their promoter regions. Additionally, we investigated the expression pattern of laccase genes in kiwifruit stems and leaves infected with Psa. Our findings revealed that laccase gene expression levels in the stems were higher than those in the leaves 5 days after inoculation with Psa. Notably, AcLAC2, AcLAC4, AcLAC17, AcLAC18, AcLAC26, and AcLAC42 showed significantly higher expression levels (p < 0.001) compared to the non-inoculated control (0 d), suggesting their potential role in resisting Psa infection. Moreover, our prediction indicated that 21 kiwifruit laccase genes are regulated by miRNA397, they could potentially act as negative regulators of lignin biosynthesis. CONCLUSIONS: These results are valuable for further analysis of the resistance function and molecular mechanism of laccases in kiwifruit.

Transcriptomics and co-expression network analysis revealing candidate genes for the laccase activity of Trametes gibbosa.

BACKGROUND: Trametes gibbosa, which is a white-rot fungus of the Polyporaceae family found in the cold temperate zone, causes spongy white rot on wood. Laccase can oxidize benzene homologs and is one of the important oxidases for white rot fungi to degrade wood. However, the pathway of laccase synthesis in white rot fungi is unknown. RESULTS: The peak value of laccase activity reached 135.75 U/min/L on the 9th day. For laccase activity and RNA-seq data, gene expression was segmented into 24 modules. Turquoise and blue modules had greater associations with laccase activity (positively 0.94 and negatively -0.86, respectively). For biology function, these genes were concentrated on the cell cycle, citrate cycle, nicotinate, and nicotinamide metabolism, succinate dehydrogenase activity, flavin adenine dinucleotide binding, and oxidoreductase activity which are highly related to the laccase synthetic pathway. Among them, gene_8826 (MW199767), gene_7458 (MW199766), gene_61 (MW199765), gene_1741 (MH257605), and gene_11087 (MK805159) were identified as central genes. CONCLUSION: Laccase activity steadily increased in wood degradation. Laccase oxidation consumes oxygen to produce hydrogen ions and water during the degradation of wood. Some of the hydrogen ions produced can be combined by Flavin adenine dinucleotide (FAD) to form reduced Flavin dinucleotide (FADH2), which can be transmitted. Also, the fungus was starved of oxygen throughout fermentation, and the NADH and FADH2 are unable to transfer hydrogen under hypoxia, resulting in the inability of NAD and FAD to regenerate and inhibit the tricarboxylic acid cycle of cells. These key hub genes related to laccase activity play important roles in the molecular mechanisms of laccase synthesis for exploring industrial excellent strains.

Substrate Specificity of LACCASE8 Facilitates Polymerization of Caffeyl Alcohol for C-Lignin Biosynthesis in the Seed Coat of Cleome hassleriana.

Catechyl lignin (C-lignin) is a linear homopolymer of caffeyl alcohol found in the seed coats of diverse plant species. Its properties make it a natural source of carbon fibers and high-value chemicals, but the mechanism of in planta polymerization of caffeyl alcohol remains unclear. In the ornamental plant Cleome hassleriana, lignin biosynthesis in the seed coat switches from guaiacyl lignin to C-lignin at ∼12 d after pollination. Here we found that the transcript profile of the laccase gene ChLAC8 parallels the accumulation of C-lignin during seed coat development. Recombinant ChLAC8 oxidizes caffeyl and sinapyl alcohols, generating their corresponding dimers or trimers in vitro, but cannot oxidize coniferyl alcohol. We propose a basis for this substrate preference based on molecular modeling/docking experiments. Suppression of ChLAC8 expression led to significantly reduced C-lignin content in the seed coats of transgenic Cleome plants. Feeding of 13C-caffeyl alcohol to the Arabidopsis (Arabidopsis thaliana) caffeic acid o-methyltransferase mutant resulted in no incorporation of 13C into C-lignin, but expressing ChLAC8 in this genetic background led to appearance of C-lignin with >40% label incorporation. These results indicate that ChLAC8 is required for C-lignin polymerization and determines lignin composition when caffeyl alcohol is available.

Application of Miscanthus substrates in the cultivation of Ganoderma lingzhi.

Ganoderma lingzhi is a traditional Chinese medicine that has been used to improve health and longevity for thousands of years. It is usually cultivated on hardwood log- or sawdust-based formulations. Conversely, in this study, we used Miscanthus sacchariflorus (MSF), M. floridulus, and M. sinensis (MSS), fast-growing perennial grasses widely distributed in China, for G. lingzhi cultivation. Mycelial growth rate, activities of lignin-degrading enzymes on colonized mushroom substrates, and expression levels of CAZymes and laccase genes based on different substrates were analyzed. Total triterpenoids, sterols, and polysaccharides content of fruiting bodies obtained from different substrates were investigated. The activities of laccase and manganese peroxidase in mycelia increased in the MSF- and MSS-based formulations compared with that in the sawdust-based formulation. The results of mycelial growth- and cultivation-related experiments showed that the Miscanthus substrates could be used as the substrates for cultivating G. lingzhi. The content of active ingredients, namely triterpenoids, sterols, and polysaccharides, in fruiting bodies cultivated on the Miscanthus substrates did not decrease compared with those in substrate obtained from the sawdust-based formulation. Therefore, the present study provides alternative substrates for the cultivation of G. lingzhi, and a reference for better utilization of inexpensive substrate in future.