Polymorphisms in Cha o 1 and Cha o 2, major allergens of Japanese cypress (Chamaecyparis obtusa) pollen from a restricted region in Japan

Japanese cedar pollinosis is a major seasonal allergy in Japan, and Japanese cypress pollinosis is a growing concern because the cypress pollen season follows the cedar pollen season and cross-reactivity among allergens occurs between these closely related species. Allergens purified from pollen under unspecified collecting conditions can potentially heterogenous allergens profiles and batch to batch variability, and amino acid sequence variants in allergens possibly exist among trees. Polymorphisms have not been investigated for the cypress pollen major allergens, Cha o 1 and Cha o 2. Our aim was to examine the homogeneity of allergen amino acid sequences. DNA sequences of Cha o 1 and Cha o 2 from pollen collected from Chiba and Ibaraki prefectures and from needles of 47 plus trees located at seed orchards in Chiba Prefecture were examined by amplicon sequencing and amino acid substitutions were deduced. Sequence analysis of the pollen samples revealed that eight and seven residues of Cha o 2 were polymorphic, respectively. Thirteen residues in Cha o 2, including those residues identified in pollen, were deduced to be polymorphic for the plus trees. Cha o 2 expressed by the 47 plus trees included amino acid differences when compared with that of isoallergen Cha o 2.0101. No substitution was deduced in Cha o 1 for pollen taken from the two prefectures. One conservative amino acid substitution was deduced in Cha o 1 for the plus trees. Of the 47 plus trees examined, 38 were deduced to express only the isoallergen Cha o 1.0101 isoform, whereas eight trees were heterozygous and a single tree was homozygous for the non-synonymous mutation, which indicates relative uniformity of Cha o 1. Cha o 2 was found to be a heterogeneous allergen which suggests that studies using pollen from different trees may not give the same results.

Chemical Structures of Adhesive and Interphase Parts in Sucrose/Citric Acid Type Adhesive Wood-Based Molding Derived from Japanese Cedar (Cryptomeria japonica)

In sucrose/citric acid based wood adhesive, the detailed bonding mechanism has still been unknown. Here, we investigated the detailed chemical structures of this adhesive wood (Japanese cedar)-based molding by using heteronuclear single quantum coherence–nuclear magnetic resonance (HSQC-NMR). NMR peaks associated with the furan-type structure appeared, suggesting that the furan compound was formed from sucrose and converted to a furan polymer during the adhesive process and that some of the furan structures in the polymers were ester-bonded with citric acid. The secondary forces between the furan polymers and wood components were thought to contribute to the adhesive effect. In our analysis of the interphase structure, primary hydroxyl groups of both polysaccharides and of lignin substructures were found to be esterified with citric acid. Additionally, some of the glycosidic bonds in polysaccharides were cleaved during the acidic condition produced by citric acid. The above results provided evidence of the polymerization of sucrose-derived 5-HMF, the esterification of wood components, and the degradation of polysaccharides during the molding process. Citric acid functioned as a clamp between the obtained furan polymer and the wood components. The sucrose/citric acid based wood adhesive can be defined as a hybrid-type wood adhesive, involving both secondary forces and chemical bonding interactions.

Production of 5-hydroxymethylfurfural from Japanese cedar (Cryptomeria japonica) in an ionic liquid, 1-methylimidazolium hydrogen sulfate

Production of 5-hydroxymethylfurfural (5-HMF) from Japanese cedar (Cryptomeria japonica) using an ionic liquid, 1-methylimidazolium hydrogen sulfate ([MIM]HSO4), was investigated. 5-HMF can be produced from C. japonica at temperatures above 120 °C. The maximum yield of 5-HMF was about 9 wt% after 15 min of treatment at 160 °C. However, 5-HMF produced in this process tended to decompose as the treatment continued. To avoid decomposition and to provide a means of recovering 5-HMF from [MIM]HSO4, three reaction systems based on [MIM]HSO4 were investigated: biphasic [MIM]HSO4/organic solvent system, [MIM]HSO4 with vacuum distillation, and [MIM]HSO4 with vacuum steam distillation. The [MIM]HSO4 reaction system combined with vacuum steam distillation was most effective. The maximum yield of 5-HMF was 17.5 wt% after treatment for 45 min at 160 °C. The combination of [MIM]HSO4 treatment with vacuum steam distillation is suitable for 5-HMF production because it is a one-pot process without the need for catalysts or pretreatment.

Plant Regeneration and In Vitro Growth Performance of Male-Sterile Somatic Plantlets of Sugi (Japanese Cedar, Cryptomeria japonica) Derived from Different Embryogenic Cell Lines

With the spread of pollinosis caused by sugi (Japanese cedar, Cryptomeria japonica) pollen, the use of pollen-free somatic seedlings of sugi is expected in Japan. However, there is a lack of knowledge on the relationship between the abilities during somatic embryogenesis, initial in vitro growth traits, and subsequent growth of somatic seedlings. In the present study, we provide the first basic information on somatic embryo maturation efficiency, somatic embryo germination, and plantlet conversion frequencies, as well as on in vitro growth performance of pollen-free somatic plantlets derived from different embryogenic cell lines (ECLs). Somatic embryo maturation efficiency varied from 34 to 514 cotyledonary embryos per plate and the average for the 19 ECLs tested was 244 embryos per plate. Subsequently, the overall average rates of somatic embryo germination and conversion among ECLs were 87.8% and 85.3%, respectively. The results of in vitro growth performance of pollen-free somatic plantlets showed significant differences in growth rate among ECLs.

Effects of a Cloth Panel Containing a Specific Ore Powder on Patients with Chamaecyparis obtusa (Cypress) Pollen Allergy

Pollen allergy to Japanese cedar and cypress is a serious illness that impairs daily life and sleep, especially during pollen season. We have reported that placing a cloth panel containing a specific natural ore powder (CCSNOP) in a room may alleviate the symptoms of hay fever and may also benefit the immune system. This ore is from the Aso mountain range, a volcano on Kyushu Island in the southwestern part of Japan. The purpose of this study was to verify the effect of CCSNOP on cypress pollen. Thirty-one double-blind tests, which investigated cedar pollen allergies, were conducted from February to March 2018 and have already been reported. After this, in early April, 10 of these cases were recruited and all had CCSNOP installed in their bedrooms. Before that, various symptoms and changes in medication were recorded in a “Symptom Diary” and included a mood survey by a questionnaire, stress test using saliva amylase, changes in cypress-specific immunoglobulins IgE and IgG4 by blood sampling, and eosinophil changes. In addition, changes in 29 types of cytokines were investigated. Exposure to CCSNOP relieved symptoms and subjects decreased their intake of medication. There was no change in mood or stress, but eosinophil levels tended to decrease. Although there were no statistical changes in cypress-specific IgE or IgG4, an increase in the former and a decrease in the latter were observed in some individuals during the period of pollen dispersal. Furthermore, levels of GM-CSF and IL8 decreased significantly after use of CCSNOP. The CCSNOP was shown to be effective against cypress pollen allergy, and future investigations will be necessary to observe the long-term effects of CCSNOP.

Marker-Assisted Selection for Pollen-Free Somatic Plants of Sugi (Japanese Cedar, Cryptomeria japonica): A Simple and Effective Methodology for Selecting Male-Sterile Mutants With ms1-1 and ms1-2

Pollen allergy caused by sugi (Japanese cedar, Cryptomeria japonica) is a serious problem in Japan. One of the measures against pollinosis is the use of male-sterile plants (MSPs; pollen-free plants). In this context, the development of a novel technique for the efficient production of sugi MSPs, which combines marker-assisted selection (MAS) with somatic embryogenesis (SE), was recently reported by our research group. To improve the efficiency of MSP production, in this paper we report improved MAS for male-sterile individuals from embryogenic cells, cotyledonary embryos, and somatic plants of sugi using a newly developed marker in the form of the causative mutation of MS1 itself, selecting individuals with ms1-1 and ms1-2 male-sterile mutations. We also describe simplified methods for extracting DNA from different plant materials and for MAS using LAMP diagnostics. Finally, we show that MAS can be efficiently performed using the one-step indel genotyping (ING) marker developed in this study and using InstaGene for DNA extraction. The combination of SE and 100% accurate marker selection during the embryogenic cell stage enables the mass production of MS1 male-sterile sugi seedlings.