Ryo Funada

Cortical microtubules (CMTs), one of the components of cytoskeleton, control the orientation and localization of newly deposited cellulose microfibrils in

ヒノキ （Chamaecyparis obtusa） は，最高品質の建材であり，日本の人工林面積の25％を占める重要な造林樹種である。持続的なヒノキ苗木の植林には，大量増殖法が必要である。本研究では，成熟種子胚由来カルスからの不定芽形成による植物体再生系の確立を目的に実験を行った。胚を2,4-ジクロロフェノキシ酢酸 （2,4-D） と6-ベンジルアミノプリン （BAP） を含む改変Campbell and Durzan培地に植え付けると，緑色カルスが誘導された。緑色カルスを暗条件から明条件へ移すと，不定芽および多芽体が形成された。多芽体からシュートを切り離して植物ホルモン無添加の培地で培養するとシュートが伸長し，その後インドール-3-酪酸 （IBA） を0，3，6 µM含む培地に移植すると全ての培地で発根が認められた。植物体を土壌に移すと正常な生育が観察され，植物体再生に成功した。

Background Cortical microtubules (CMTs), one of the components of cytoskeleton, control the orientation and localization of newly deposited cellulose microfibrils in cell walls, and thereby determine the shape, size, and structure of plant cells. Imaging of CMTs in plant tissues is generally performed using fluorescently labeled specimens under an optical fluorescence or confocal laser scanning microscope. However, optical microscopy has insufficient resolution to visualize individual CMTs, and its observation range is limited to superficial tissue layers that light can penetrate. In contrast, transmission electron microscopy offers high-resolution visualization of CMTs in plant cells but is restricted to slightly oblique ultrathin sections with an approximate thickness of 70–100 nm. Results Herein, we introduce a technique for visualizing CMTs within unstained plant tissues by combining cryofracture techniques with field emission scanning electron microscopy (FE-SEM). We successfully observed the arrangement of CMTs in several plant specimens, including young branches of ginkgo ( Ginkgo biloba ), calli from the leaves of hybrid poplar ( Populus sieboldii × P. grandidentata ), and root tips of the adzuki bean ( Vigna angularis ). CMTs were visualized on the protoplasmic fracture face using both cryo-FE-SEM and conventional room-temperature FE-SEM. Conclusions The combination of freeze-fracture techniques with FE-SEM enables the visualization of CMT arrangement in plant tissues at a high resolution and across a broad area without the need for staining or extraction of cellular components. This technique is applicable to various plant tissues and allows for detailed observation of CMTs within these tissues, providing valuable insights into the role of microtubules in the division and differentiation of plant cells.

Background and Aims: Xylem and vessel structures help to maintain water transport in woody plants in response to environmental changes. Many studies have demonstrated the relationships between vessel structures and, in particular, temperature. However, the effects of environmental factors on the vessel size distribution and on vessels of different sizes have not been fully assessed statistically. Lianas are characterized by large vessels and vessel dimorphism. Liana abundance decreases with decreasing temperature; this pattern is attributed to the vulnerability of their large vessels to freeze–thaw embolism. However, in temperate zones, the relationships between liana abundance and temperature differ between twining and root climbers. Methods: We sampled wood discs of eight liana species distributed across Japan and measured the size and shape of 130 940 vessels in 836 sections from 219 individuals. We classified vessels of each species into two diameter clusters (large and small) and calculated vessel traits and potential hydraulic conductivity (Kp). Vessel traits were compared among climbing mechanisms, and the relationships between vessel traits and temperature were analysed for species. Key Results: Twining climbers had larger vessel diameters than root climbers and had greater Kp. However, the relationships between temperature and vessel traits of species were inconsistent within climbing mechanisms. The decrease in Kp in certain species with decreasing temperature might result from species-specific changes in xylem structure. Vessels of the two clusters related differently to temperature in some species. Conclusions: The vessel traits in each climbing mechanism might partly explain the distribution patterns of these lianas in the study region. Furthermore, changes in Kp in some species supported the prediction that liana competitiveness decreases with decreasing temperature. Understanding the mechanisms behind the changes in vessel traits and vessel size distribution along environmental factors will provide fundamental insights into how environmental changes affect forest ecosystems by altering plant hydraulic function.

Angiosperm trees develop tension wood on the upper side of leaning stems to reorient the direction of stems towards their normal positions in response to a gravitational stimulus. The development of gelatinous fibers with thick inner gelatinous layers (G-layers) might elevate tensile stress required for such reorientation in many angiosperm species. This study aims to investigate negative gravitropism and tension wood formation in response to a gravitational stimulus in tree species with different respective wood densities. Seedlings of three tropical trees, namely, Diospyros celebica, Artocarpus heterophyllus, and Falcataria moluccana were artificially inclined at 45° from the vertical and harvested three months later for analysis of plant gravitropism and tension wood formation. Inclined seedlings of the three species exhibited different rates of stem recovery and movement towards the vertical. The widths of region of tension wood in the thickness of G-layers were positively correlated with the negative gravitropism of stems. However, such relationships differed significantly among the three species. The differences in patterns of negative gravitropism of stems, widths of tension wood and thicknesses of G-layers in inclined seedlings of F. moluccana, A. heterophyllus, and D. celebica were due to differences among species rather than to differences in the wood density of the respective species. Larger amounts of gelatinous fibers and/or thicker G-layers were essential for the negative gravitropism of inclined stems. However, each tree species exhibited different features during stem recovery.

Assessing the effects of air pollutants, including aerosols, on trees is important for protecting forests in the future. This study determined the adsorption of particles on trees after 1- or 2-year long-term exposure (for 1 or 2 h/day) to submicron-scale ammonium sulfate (AS) particles using a field-emission scanning electron microscope (FE-SEM). Energy-dispersive X-ray spectroscopy (EDX) was also used to distinguish particles resulting from exposure from those present on the leaves under natural conditions prior to the 1- or 2-year exposure. We found submicron-sized AS particles were deposited on the leaf surfaces of four tree species after long-term exposure in a growth chamber < 70% humidity. These particles occurred as individual deposits without aggregation on the abaxial and adaxial surfaces. The particle shape deposited on the leaf surface in short-term (3–30 min) exposures in a growth chamber < 70% humidity was spherical with no corners, whereas that in long-term exposures was nonspherical flattened, angular, or irregular. Few micrometers was also observed, differing from 300 to 600 nm in diameter at exposure. These differences could be caused by the possibility that the particles have been deposited for a long time or that the humidity on the leaf surface has caused them to deliquescence and change shape after deposition. We hypothesized that these particle changes facilitate the uptake of AS into the leaf interior.

Resin canals serve as a natural feature with the function of a defense system against fungi, bacteria, and insects. Trees can form these canals in response to mechanical injury and ecological disturbance. Factors, such as plant hormones and temperature, influence cambial activity and cell differentiation. This study examined the effects of increased temperature and plant hormones on cambial reactivation, vessel formation, and resin canal formation using localized heating and the application of the ethylene generator ethephon to dormant stems of the Toxicodendron vernicifluum seedlings. Localized heating was achieved by wrapping an electric heating ribbon around dormant stems, while ethephon was applied to the bark surface. Treatment was initiated on 29 January 2021, including control, heating, ethephon, and a combination of heating and ethephon. Cambial reactivation and resin canal formation were monitored using light microscopy, and bud growth was recorded with a digital camera. Localized heating induced earlier phloem reactivation, cambial reactivation, and xylem differentiation, increasing the number of vessels. The application of exogenous ethylene delayed these processes. The combination of localized heating and exogenous ethylene application resulted in smaller vessels and larger resin canals. These results suggest that increased temperature plays a significant role in cambial reactivation and vessel formation in ring-porous hardwood and that ethylene affects vessel differentiation and resin canal development.

オオヤマレンゲ（Magnolia sieboldii subsp. japonica）は，日本原産の落葉樹木である。複数の県で絶滅危惧I類またはII類に指定されており，希少価値の高い樹種である。本研究では，オオヤマレンゲの植物体再生系の確立を目標として，未成熟種子をα-ナフタレン酢酸と6-ベンジルアミノプリンを添加したLepoivre 培地，窒素源を半分にしたMurashige and Skoog培地およびWoody Plant Medium培地に植え付けた。その結果，不定胚形成細胞（Proembryogenic masses; PEMs）の誘導と不定胚の形成が認められた。PEMsの誘導には，開花から1～3週間後に採取した未成熟種子が適していた。不定胚の一部は明条件下で植物体に成長し，組織培養を用いた未成熟種子からの植物体再生に成功した。

Water stress has a significant impact on tree growth. However, the effects of watering on cambial activity and its influence on tree growth in subtropical climates is poorly understood. The present study analyzed the cambial activity on the stem of evergreen hardwood Samanea saman in response to either high frequency or low frequency watering during the pre-monsoon season in subtropical Bangladesh. We used two groups of seedlings: one group of seedlings was watered daily (high frequency watering), while the second group of seedlings was watered at 4–5-day intervals (low frequency watering). Samples for sequential observations of cambial activity by microscopy were collected from the main stems of seedlings of both groups. At the start of the experiment on March 25, 2015, during the pre-monsoon season, the cambium was inactive with no evidence of cell division. After 10 days of high frequency watering, cambial cell division and xylem differentiation were initiated. New cell plates were formed in the phloem side of the cambium. However, the cambium was inactive when low frequency watering was supplied. Supplying water in high frequency reactivated the cambium with forming small to large vessels. In contrast, the cambium remained inactive when low frequency watering was supplied throughout the experiment. These results suggest that continuous supply of water to the soil is one of the most important factors for cambial reactivation during pre-monsoon season in subtropical trees. Furthermore, our findings of artificial watering treatments might help to better understand the response of cambium to changes in precipitation patterns under natural conditions, allowing us to learn more about how cambium of subtropical trees responds to climate change.

The secondary tissues of woody plants consist of fragile cells and rigid cell walls. However, the structures are easily damaged during mechanical cross-sectioning for electron microscopy analysis. Broad argon ion beam (BIB) milling is commonly employed for scanning electron microscopy (SEM) of hard materials to generate a large and distortion-free cross-section. However, BIB milling has rarely been used in plant science. In the present study, SEM combined with BIB milling was validated as an accurate tool for structural observation of secondary woody tissues of two samples, living pine (Pinus densiflora) and high-density oak wood (Quercus phillyraeoides), and compared with classical microtome cross-sectioning. The BIB milling method does not require epoxy resin embedding because of prior chemical fixation and critical point drying of the sample, thus producing a three-dimensional image. The results showed that xylem structures were well-preserved in their natural state in the BIB-milled cross-section compared with the microtome cross-section. The observations using SEM combined with BIB milling were useful for wide-area imaging of both hard and soft plant tissues, which are difficult to observe with transmitted electron microscopy because it is difficult to obtain sections of such tissues, particularly those of fragile reaction woods.