BEL1-like Homeodomain Protein BLH6a Is a Negative Regulator of CAl5H2 in Sinapyl Alcohol Monolignol Biosynthesis in Poplar

Lignin is one of the major components of xylem cell walls in tree stems. The lignin in the wood of most flowering plants (dicotyledonous angiosperms) is typically polymerized from three monolignol precursors, coniferyl alcohol, sinapyl alcohol, and p-coumaroyl alcohol, resulting in guaiacyl (G), syringyl (S), and hydroxyphenyl (H) subunits, respectively. In this study, we focus on the transcriptional regulation of a coniferaldehyde 5-hydroxylase (CAld5H2) gene, which encodes a key enzyme for sinapyl alcohol biosynthesis. We carried out a yeast one-hybrid (Y1H) screen to identify candidate upstream transcription factors (TFs) regulating CAld5H2. We obtained 12 upstream TFs as potential regulators of CAld5H2. One of these TF genes, BLH6a, encodes a BEL1-like homeodomain (BLH) protein and negatively regulated the CAld5H2 promoter activity. The direct regulation of CAld5H2 promoter by BLH6a was supported by chromatin immunoprecipitation–quantitative polymerase chain reaction (ChIP–qPCR) and dominant repression of BLH6a in transgenic plants. Luciferase complementation imaging analyses showed extensive protein–protein interactions among these 12 TFs. We propose that BLH6a is a negative regulator of CAld5H2, which acts through combinatorial regulation of multiple TFs for sinapyl alcohol (S monolignol) biosynthesis in poplar.

Caffeine Interactions with Wood Polymers

Caffeine is a verified bioactive substance suitable for wood protection against pests. Unlike studies of the biocidal effects of caffeine, caffeine-wood bonds and interactions with wood polymer structures have not been studied whatsoever thus far. For this reason, caffeine (1 g/L) interactions with the main wood components (cellulose; hemicellulose; lignin and its precursors conipheryl alcohol, sinapyl alcohol, coumaryl alcohol) were analyzed in the present study. Caffeine concentrations were analyzed using UV–VIS spectrometry at wavelength 287 nm. The results confirmed caffeine variable binding with wood components in comparison to controls (pure caffeine). Cellulose and sinapyl alcohol did not interact with caffeine. Caffeine was bonded with the rest of the wood components in an increasing rank: conipheryl alcohol = lignin < hemicellulose < coumaryl alcohol. These results have a significant role in the protection of wood depending on its chemical composition and the wood species.

EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification

Bioorthogonal chemical reporter strategy can be successfully combined with EPR spectroscopy in plant tissues to detect the incorporation of a tagged monolignol into the lignin polymer.

Correction: EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification

Correction for ‘EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification’ by Clémence Simon et al., Chem. Commun., 2021, DOI: 10.1039/D0CC05218C.

Subcritical Methanol Extraction of the Stone of Japanese Apricot Prunus mume Sieb. et Zucc.

The pits of Japanese apricot, Prunus mume Sieb. et Zucc., which are composed of stones, husks, kernels, and seeds, are unused by-products of the processing industry in Japan. The processing of Japanese apricot fruits generates huge amounts of waste pits, which are disposed of in landfills or, to a lesser extent, burned to form charcoal. Mume stones mainly consist of cellulose, hemicellulose, and lignin. Herein, we attempted to solubilize the wood-like carapace (stone) encasing the pit by subcritical fluid extraction with the aim of extracting useful chemicals. The characteristics of the main phenolic constituents were elucidated by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) analyses. The degrees of solubility for various treatments (190 °C; 3 h) were determined as follows: subcritical water (54.9%), subcritical 50% methanol (65.5%), subcritical 90% methanol (37.6%), subcritical methanol (23.6%), and subcritical isopropyl alcohol (14.4%). Syringaldehyde, sinapyl alcohol, coniferyl alcohol methyl ether, sinapyl alcohol methyl ether, 5-(hydroxymethyl)-2-furfural, and furfural were present in the subcritical 90% methanol extract. Coniferyl and sinapyl alcohols (monolignols) are source materials for the biosynthesis of lignin, and syringaldehyde occur in trace amounts in wood. Our current findings provide a solubilization method that allows the main phenolic constituents of the pits to be extracted under mild conditions. This technique for obtaining subcritical extracts shows great potential for further applications.

Inhibition by Substrates of a Coniferyl Alcohol Dehydrogenase Purified from Sugarcane Stalks

Aims and Objectives: This study aimed to characterize a coniferyl alcohol dehydrogenase from sugarcane stalks. Also, the purification of CAD from sugarcane stalks was also carried out to study kinetic properties and substrate specificity. Background: Sugarcane plants contain an alcohol dehydrogenase able to reduce both coniferyl and sinapyl aldehydes to their correspondent alcohols, although there are reasonable grounds for suspecting that these are two distinct enzymes. Methods: The enzyme, coniferyl alcohol dehydrogenase was 125-fold purified from sugarcane stalks. Its activity was estimated by HPLC by calculating the amount of product formed. Results: The enzyme showed an optimum pH value of 7.9, at an optimum temperature of 20-22°C and a molecular mass of 48 kDa. The Km value for coniferyl alcohol was 3.03 µM and the enzyme was shown to be inhibited by an excess of the substrate from 17 µM. This dehydrogenase showed a similar affinity to sinapyl alcohol (Km 1.78 µM). Conclusions: This paper provides circumstantial evidence about the existence of two different alcohol dehydrogenases, specific to each of the substrates.

Enabling microbial syringol conversion through structure-guided protein engineering

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism isO-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringolO-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover inPseudomonas putidaKT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromaticO-demethylases in the biological conversion of lignin-derived aromatic compounds.