Artificially lignified cell wall catalyzed by peroxidase selectively localized on a network of microfibrils from cultured cells.

MAIN CONCLUSION: An artificial lignified cell wall was synthesized in three steps: (1) isolation of microfibrillar network; (2) localization of peroxidase through immunoreaction; and (3) polymerization of DHP to lignify the cell wall. Artificial woody cell wall synthesis was performed following the three steps along with the actual formation in nature using cellulose microfibrils extracted from callus derived from Cryptomeria japonica. First, we constructed a polysaccharide network on a transmission electron microscopy (TEM) grid. The preparation method was optimized by chemical treatment, followed by mechanical fibrillation to create a microfibrillated network. Morphology was examined by TEM, and chemical characterization was by Fourier transform infrared (FTIR) spectroscopy. Second, we optimized the process to place peroxidase on the microfibrils via an immunoreaction technique. Using a xyloglucan antibody, we could ensure that gold particles attached to the secondary antibodies were widely and uniformly localized along with the microfibril network. Third, we applied the peroxidase attached to secondary antibodies and started to polymerize the lignin on the grid by simultaneously adding coniferyl alcohol and hydrogen peroxide. After 30 min of artificial lignification, TEM observation showed that lignin-like substances were deposited on the polysaccharide network. In addition, FTIR spectra revealed that the bands specific for lignin had increased, demonstrating the successful artificial formation of woody cell walls. This approach may be useful for studying woody cell wall formation and for producing made-to-order biomaterials.

Cloning and Functional Analysis of Lignin Biosynthesis Genes Cf4CL and CfCCoAOMT in Cryptomeria fortunei.

Cryptomeria fortunei, also known as the Chinese cedar, is an important timber species in southern China. The primary component of its woody tissues is lignin, mainly present in secondary cell walls. Therefore, continuous lignin synthesis is crucial for wood formation. In this study, we aimed to discover key genes involved in lignin synthesis expressed in the vascular cambium of C. fortunei. Through transcriptome sequencing, we detected expression of two genes, 4CL and CCoAOMT, known to be homologous to enzymes involved in the lignin synthesis pathway. We studied the function of these genes through bioinformatics analysis, cloning, vascular cambium expression analysis, and transgenic cross-species functional validation studies. Our results show that Cf4CL and CfCCoAOMT do indeed function in the pathway of lignin synthesis and likely perform this function in C. fortunei. They are prime candidates for future (gene-editing) studies aimed at optimizing C. fortunei wood production.

Human cystatin SN is an endogenous protease inhibitor that prevents allergic rhinitis.

BACKGROUND: Protease allergens disrupt epithelial barriers to exert their allergenicity. Cystatin SN (encoded by CST1) is an endogenous cysteine protease inhibitor upregulated in nasal epithelia in patients with allergic rhinitis (AR). OBJECTIVE: We sought to investigate the protective effect of human cystatin SN on AR symptoms using pollen-induced AR mouse models. METHODS: We performed an in vitro protease activity assay to evaluate the effect of recombinant human cystatin SN (rhCystatin SN) on Japanese cedar (JC) or ragweed proteases. A human nasal epithelial cell line, RPMI 2650, was used to examine tight junction (TJ) disruption in vitro. Mice were sensitized and nasally challenged with JC or ragweed pollens with or without rhCystatin SN to examine the effect of rhCystatin SN on AR symptoms and the epithelial barrier in vivo. Because mice lack CST1, we generated transgenic (Tg) mice expressing human CST1 under control of its genomic control region (hCST1-Tg mice) to examine the role of cystatin SN in physiologically expressed conditions. RESULTS: rhCystatin SN inhibited JC but not ragweed protease activities and prevented JC-induced but not ragweed-induced TJ disruption in vitro. Exogenous administration of rhCystatin SN ameliorated JC-induced but not ragweed-induced sneezing and nasal TJ disruption in vivo. Furthermore, hCST1-Tg mice showed decreased JC-induced but not ragweed-induced sneezing symptoms and nasal TJ disruption compared with wild-type mice. CONCLUSION: Human cystatin SN suppresses AR symptoms through inhibiting allergen protease activities and protecting the nasal TJ barrier in an allergen-specific manner. We propose that upregulation of nasal endogenous protease inhibitors, including cystatin SN, is a novel therapeutic strategy for protease allergen-induced AR.

Structure and Enzymatic Properties of a Two-Domain Family GH19 Chitinase from Japanese Cedar ( Cryptomeria japonica) Pollen.

CJP-4 is an allergen found in pollen of the Japanese cedar Cryptomeria japonica. The protein is a two-domain family GH19 (class IV) Chitinase consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. Here, we produced recombinant CJP-4 and CBM18-truncated CJP-4 (CJP-4-Cat) proteins. In addition to solving the crystal structure of CJP-4-Cat by X-ray crystallography, we analyzed the ability of both proteins to hydrolyze chitin oligosaccharides, (GlcNAc) n, polysaccharide substrates, glycol chitin, and ß-chitin nanofiber and examined their inhibitory activity toward fungal growth. Truncation of the CBM18 domain did not significantly affect the mode of (GlcNAc) n hydrolysis. However, significant effects were observed when we used the polysaccharide substrates. The activity of CJP-4 toward the soluble substrate, glycol chitin, was lower than that of CJP-4-Cat. In contrast, CJP-4 exhibited higher activity toward ß-chitin nanofiber, an insoluble substrate, than did CJP-4-Cat. Fungal growth was strongly inhibited by CJP-4 but not by CJP-4-Cat. These results indicate that the CBM18 domain assists the hydrolysis of insoluble substrate and the antifungal action of CJP-4-Cat by binding to chitin. CJP-4-Cat was found to have only two loops (loops I and III), as reported for ChiA, an allergenic class IV Chitinase from maize.

Enzymatic Specific Production and Chemical Functionalization of Phenylpropanone Platform Monomers from Lignin.

Enzymatic catalysis is an ecofriendly strategy for the production of high-value low-molecular-weight aromatic compounds from lignin. Although well-definable aromatic monomers have been obtained from synthetic lignin-model dimers, enzymatic-selective synthesis of platform monomers from natural lignin has not been accomplished. In this study, we successfully achieved highly specific synthesis of aromatic monomers with a phenylpropane structure directly from natural lignin using a cascade reaction of ß-O-4-cleaving bacterial enzymes in one pot. Guaiacylhydroxylpropanone (GHP) and the GHP/syringylhydroxylpropanone (SHP) mixture are exclusive monomers from lignin isolated from softwood (Cryptomeria japonica) and hardwood (Eucalyptus globulus). The intermediate products in the enzymatic reactions show the capacity to accommodate highly heterologous substrates at the substrate-binding sites of the enzymes. To demonstrate the applicability of GHP as a platform chemical for bio-based industries, we chemically generate value-added GHP derivatives for bio-based polymers. Together with these chemical conversions for the valorization of lignin-derived phenylpropanone monomers, the specific and enzymatic production of the monomers directly from natural lignin is expected to provide a new stream in "white biotechnology" for sustainable biorefineries.

NMR assignments and ligand-binding studies on a two-domain family GH19 chitinase allergen from Japanese cedar (Cryptomeria japonica) pollen.

A two-domain family GH19 chitinase from Japanese cedar (Cryptomeria japonica) pollen, CJP-4, which consists of an N-terminal CBM18 domain and a GH19 catalytic domain, is known to be an important allergen, that causes pollinosis. We report here the resonance assignments of the NMR spectrum of CJP-4. The backbone resonances were almost completely assigned, and the secondary structure was estimated based on the chemical shift values. The addition of a chitin dimer to the enzyme solution perturbed the chemical shifts of the resonances of amino acid residues forming a long extended binding site spanning from the CBM18 domain to the GH19 catalytic domain.

NADPH oxidase activity in allergenic pollen grains of different plant species.

Pollen is an important trigger of allergic diseases. Recent studies have shown that ragweed pollen NAD(P)H oxidase generates reactive oxygen species (ROS) and plays a prominent role in the pathogenesis of allergies in mouse models. Here, we demonstrated that allergenic pollen grains showed NAD(P)H oxidase activity that differed in intensity and localization according to the plant families. The activity occurred at the surface or in the cytoplasm in pollen of grasses, birch, and ragweed; in subpollen particles released from ragweed pollen; and at the inner surface or in the cytoplasm but not on the outer wall, which was sloughed off after the rupture, of pollen of Japanese cedar and Japanese cypress. The activity was mostly concentrated within insoluble fractions, suggesting that it facilitates the exposure of tissues to ROS generated by this enzyme. The extent of exposure to pollen-generated ROS could differ among the plant families.

A frameshift mutation of the chloroplast matK coding region is associated with chlorophyll deficiency in the Cryptomeria japonica virescent mutant Wogon-Sugi.

Wogon-Sugi has been reported as a cytoplasmically inherited virescent mutant selected from a horticultural variety of Cryptomeria japonica. Although previous studies of plastid structure and inheritance indicated that at least some mutations are encoded by the chloroplast genome, the causative gene responsible for the primary chlorophyll deficiency in Wogon-Sugi, has not been identified. In this study, we identified this gene by genomic sequencing of chloroplast DNA and genetic analysis. Chloroplast DNA sequencing of 16 wild-type and 16 Wogon-Sugi plants showed a 19-bp insertional sequence in the matK coding region in the Wogon-Sugi. This insertion disrupted the matK reading frame. Although an indel mutation in the ycf1 and ycf2 coding region was detected in Wogon-Sugi, sequence variations similar to that of Wogon-Sugi were also detected in several wild-type lines, and they maintained the reading frame. Genetic analysis of the 19 bp insertional mutation in the matK coding region showed that it was found only in the chlorophyll-deficient sector of 125 full-sibling seedlings. Therefore, the 19-bp insertion in the matK coding region is the most likely candidate at present for a mutation underlying the Wogon-Sugi phenotype.

Expressed sequence tags from Cryptomeria japonica sapwood during the drying process.

Secondary metabolites called norlignans are produced in the xylem of Cryptomeria japonica D. Don. Several norlignans have roles in the defense of sapwood against microbial invasion and in the coloration of heartwood. The biosynthetic pathway of norlignans is largely unknown. Norlignans have been reported to accumulate in the sapwood during the drying of C. japonica logs. To search for genes encoding enzymes that catalyze the synthesis of norlignans, we carried out suppression subtractive hybridization using the fresh sapwood of a felled log and the drying sapwood in which a norlignan, agatharesinol, accumulated. A total of 1050 expressed sequence tags were obtained from the subtracted cDNA library, and these were assembled into 146 contigs and 361 singletons. Of these 507 unique sequences, 263 were functionally classified into 12 categories. "Metabolism" was the largest category, with 23% (61) of classified sequences. Twenty-six sequences that encode 16 enzymes were assigned to "secondary metabolism." Expression analysis of 15 genes related to "secondary metabolism" revealed that 12 of these genes had transcripts that were induced during the sapwood drying process. Of the 12 genes, 10 encoded enzymes that use aromatic compounds as substrates. In addition, 58 sequences representing 22 defense-related proteins were found. Our subtraction library should be a useful source for isolating genes encoding proteins involved in secondary metabolism including norlignan biosynthesis and defense in C. japonica xylem.

Characteristics of an aminopeptidase from Japanese cedar (Cryptomeria japonica) pollen.

A peptidase from Japanese cedar pollen, Jc-peptidase, was clarified to preferentially hydrolyze an MCA substrate of Phe-MCA (L-phenylalanyl-4-methylcoumaryl-7-amide). This study examined substrate specificities of Jc-peptidase using oligopeptides. Jc-peptidase hydrolyzed Phe-Phe and Tyr-Phe effectively and hydrolyzed Leu-Phe, Met-Phe, and Arg-Phe moderately. Other substrates such as Ala-Phe, Asp-Phe, and Pro-Phe were not hydrolyzed with the peptidase. Results obtained with a series of aminoacyl-Phe peptides were compatible with the facts obtained for MCA substrates except for Arg-MCA. Effects of amino acid residues in the P1' position were also examined using Phe-amino acids. An N-terminal phenylalanine residue was actually released from bioactive peptides such as molluscan cardioexcitatory neuropeptide (FMRF-NH(2)). Because the activity was inhibited with Zn(2+) and EDTA, Jc-peptidase was inferred to belong to the metalloproteases. The N-terminal amino acid sequence was determined to be APIGVQLEIEENYVHMYNGF and an internal sequence to be EIFAATFNVDEETEA, but no homology with other proteins was found.

A heartwood norlignan, (E)-hinokiresinol, is formed from 4-coumaryl 4-coumarate by a Cryptomeria japonica enzyme preparation.

An enzyme preparation from the cultured cells of Cryptomeria japonica catalyses the formation of a heartwood norlignan, (E)-hinokiresinol, from two distinct phenylpropanoid monomers: 4-coumaroyl CoA and 4-coumaryl alcohol, and from a dimer: 4-coumaryl 4-coumarate.