Infrared (IR) microspectroscopy combined with a quartz crystal microbalance (QCM) together with an original relative humidity (RH) control system has been developed for studying water adsorption on a collagen film. The adsorbed water weights measured by QCM are almost similar for wetting and drying processes at 28 ℃, indicating that the collagen film is close to the water adsorption/desorption equilibria. A broad OH + NH stretching band area (3000-3700 cm) in the IR spectra of the collagen film increased linearly with the adsorbed weight until about 1.2 μg/8.0 μg dry collagen film at relative humidity (RH) = 40%, while at higher RH (60%, 80%), the band area deviates from the linear trend to the lower side, due to viscoelasticity and others. The OH + NH band can be simulated by four Gaussian components at 3440, 3330, 3210, and 3070 cm with the relatively constant band areas of 3330 and 3070 cm components due to amide A and B (NH) for increasing and decreasing RH. Bound water (3210 cm component: short H bond) constitutes around 70% of total water (3440 + 3210 cm band areas) at RH = 4.9% but decreases to 23% at RH = 80.3%, where free water (3440 cm component: long H bond) becomes dominant over 70%. The peak shifts of C=O stretching (Amide I) and N-H bending (Amide II) can be understood by increasing hydrogen bonding of water molecules (bound water) bound to peptides at lower RH. The higher wavenumber shifts of CH stretching can be due to the loose binding of water molecules (free water) to aliphatic chains on the collagen surface, especially at higher RH. The present combined QCM-IR method is useful for studying amounts and natures of water adsorbing on biomolecules.

Recent studies in the tallest tree species suggest that physiological and anatomical traits of tree-top leaves are adapted to water-limited conditions. In order to examine water retention mechanism of leaves in a tall tree, infrared (IR) micro-spectroscopy was conducted on mature leaf cross-sections of tall Cryptomeria japonica D. Don from four different heights (51, 43, 31 and 19 m). We measured IR transmission spectra and mainly analyzed OH (3700-3000 cm-1) and C-O (1190-845 cm-1) absorption bands, indicating water molecules and sugar groups, respectively. The changes in IR spectra of leaf sections from different heights were compared with bulk-leaf hydraulics. Both average OH band area of the leaf sections and leaf water content were larger in the upper-crown, while osmotic potential at saturation did not vary with height, suggesting higher dissolved sugar contents of upper-crown leaves. As cell-wall is the main cellular structure of leaves, we inferred that larger average C-O band area of upper-crown leaves reflected higher content of structural polysaccharides such as cellulose, hemicellulose and pectin. Infrared micro-spectroscopic imaging showed that the OH and C-O band areas are large in the vascular bundle, transfusion tissue and epidermis. Infrared spectra of individual tissue showed that much more water is retained in vascular bundle and transfusion tissue than mesophyll. These results demonstrate that IR micro-spectroscopy is a powerful tool for visualizing detailed, quantitative information on the spatial distribution of chemical substances within plant tissues, which cannot be done using conventional methods like histochemical staining. The OH band could be well reproduced by four Gaussian OH components around 3530 (free water: long H bond), 3410 (pectin-like OH species), 3310 (cellulose-like OH species) and 3210 (bound water: short H bond) cm-1, and all of these OH components were higher in the upper crown while their relative proportions did not vary with height. Based on the spectral analyses, we inferred that polysaccharides play a key role in biomolecular retention of water in leaves of tall C. japonica.

To examine differences of water-retention mechanisms between pectins with and without Ca, quartz crystal microbalance (QCM) and infrared microspectroscopy combined with a humidity-control system were used to analyze differences in amounts and species of adsorbed water to pectins without and with Ca. QCM analysis shows that water contents are ∼2-3 times larger for the pectin film with Ca than that without Ca. The difference IR spectra suggest that long, medium, and short H-bond water molecules (free, medium, and bound water) are adsorbed to the pectin film without Ca. IR peak shifts of C═O of COOH and C-OH suggest that these water molecules are hydrogen-bonded to C═O and C-OH groups. In addition to these water molecules, bulk water is adsorbed. IR OH band areas fitted by four Gaussian components show that bulk water is mainly adsorbed to the pectin film with Ca, possibly among skeletal chains of pectin bridged by Ca.

Restoration of species-rich semi-natural grassland requires not only a seed source but also appropriate soil properties. In Europe, approximately 10 years are required for the properties of fertilized soils to reach suitable conditions and be considered successfully restored. However, restoration may require additional time in Japan because heavier precipitation causes leaching of basic cations from soils, resulting in soil acidification; volcanic ejecta also forms active Al and Fe hydroxides with high phosphate sorption. Within this context, we aimed to answer the following questions: i) whether and how the impacts of fertilization remain in the soil properties after half a century in Japan; and ii) how fertilization affects the restoration of semi-natural grasslands in Japan. We investigated the vegetation and soil properties of a Zoysia japonica pasture improved half a century ago with a single application of fertilizer and an adjacent semi-natural grassland (native pasture) in Japan, and found the following: (1) the two pastures had similar dominance of Z. japonica, but differed in the species composition; (2) the improved pasture exhibited lower species richness than the native pasture; (3) soil nutrients, including N, P, K, Mg, and Ca, were higher in the improved pasture than in the native pasture; and (4) many chemical properties of the soils were associated with species composition; namely, the vegetation on nutrient-rich soil had more alien species and fewer native species. We conclude that a single dose of fertilization can affect soil properties in semi-natural grasslands over half a century in Japan, leading to species loss and changing the species composition. We suggest that fertilized soils under grazing in Japan may require more than half a century to restore the nutrients to suitable levels for the establishment of a species-diverse grassland.