Conducting Pathways in North Temperate Deciduous Broadleaved Trees

IAWA Journal, Vol. 29 (3), 2008: 247–263 CONDUCTING PATHWAYS IN NORTH TEMPERATE DECIDUOUS BROADLEAVED TREES Toshihiro Umebayashi1, Yasuhiro Utsumi2,*, Shinya Koga2, Susumu Inoue2, Seizo Fujikawa3, Keita Arakawa3, Junji Matsumura4 and Kazuyuki Oda4 SUMMARY The interspecific variation of dye ascent in the stems of 44 broadleaved deciduous species growing in Japan was studied using freeze-dried samples after dye injection. The dye ascending pattern differed both within and between ring-porous and diffuse-porous species. In large earlywood vessels of all ring-porous species, the dye ascended only in the outermost annual ring, and the inner annual rings had lost their water transport function. The dye ascending pattern within the inner annual rings in the ring-porous species was categorized into three types: i) the dye ascended both in the many latewood vessels throughout the latewood and small earlywood vessels; ii) the dye ascended in the many vessels throughout the latewood; and iii) the dye ascended mainly in the late latewood vessels. In diffuse-porous species, the dye ascending pattern within the annual rings also was categorized into three types: i) the dye ascended throughout the annual rings; ii) the dye ascended mainly in the earlywood vessels; and iii) the dye ascended mainly in the latewood vessels. Xylem water distribution was also examined by cryo-SEM in three ring-porous and three diffuse-porous species that had different dye ascending patterns. The water distribution pattern within annual rings was correlated with the dye ascending pattern except for one diffuse- porous species (Salix gracilistyla). In this case, water was distributed in the whole region of the annual rings although dye was mainly distributed in the earlywood. These results showed that the functional area of water transport within annual rings differed among ring-porous species and diffuse-porous species. Key words: Dye, cavitation, diffuse-porous, ring-porous, deciduous trees. 1) Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki 812-8581, Japan. 2) Shiiba Research Forest, Graduate School of Agriculture, Kyushu University, Shiiba 883-0402, Japan. 3) Laboratory of Wood Biology, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. 4) Laboratory of Wood Science, Faculty of Agriculture, Kyushu University, Hakozaki 812-8581, Japan. *) Corresponding author [E-mail: [email protected]]. Associate Editor: John Sperry Downloaded from Brill.com10/05/2021 06:51:38AM via free access 248 IAWA Journal, Vol. 29 (3), 2008 Umebayashi et al. — Conducting pathways in temperate trees 249 INTRODUCTION Transpiring woody plants move the water from the soil to the leaves through the conduits (vessels and tracheids) of their stems, and the anatomical characteristics of the stem water-conducting pathway can have a profound impact on the efficiency of hydraulic conductivity (Tyree & Zimmermann 2002). In stems of broadleaved trees growing in northern temperate zones, ring-porous species have considerably larger diameter vessels in the earlywood, and the earlywood vessels in the outermost annual ring have a major role in water transport (Chaney & Kozlowski 1977; Ellmore & Ewers 1986; Granier et al. 1994). On the other hand, diffuse-porous species have similar diameter vessels scattered throughout the whole region of an annual ring, and the vessels in the outer several annual rings have a role in water transport (Baker & James 1933; Kozlowski & Winget 1963; Anfodillo et al. 1993). These results indicate that the functional region for water transport in the stem xylem is obviously different between ring-porous trees and diffuse-porous trees. However, in broadleaved trees, there are many ring-porous and diffuse-porous species and their xylem structures vary widely (Wheeler et al. 1989; Carl- quist 2001). The interspecific variation of the water transport pathways in ring-porous and diffuse-porous species is not fully understood. To evaluate the xylem sap flow of the stems of transpiring woody plants, various methods have been used. Thermal methods such as the heat pulse method (Swanson 1994) and the thermal dissipation method (Granier 1987; Lu et al. 2004) have revealed the radial profile of sap flow in the trunk (Čermáket al. 1992; James et al. 2002). Tracer injection methods such as dye injection (Baker & James 1933; Koslowski et al. 1965; Sakamoto & Sano 2000; Kuroda 2005), radioactive isotope injection (Postlethwait & Rogers 1958; Thomas 1967), and deuterium injection (Meinzer et al. 2003, 2006) have been used to describe the sap pathway. To visualize water distribution in the xylem, nuclear magnetic resonance imaging (Holbrook et al. 2001; Clearwater & Clark 2003; Utsuzawa et al. 2005) and cryo-scanning electron microscopy (cryo-SEM, Ohtani & Fujikawa 1990; Utsumi et al. 1996, 2003) have also been used. In the various methods mentioned above, the dye injection method is relatively easy to conduct and has been widely used to visualize the water-conducting pathway at the tissue level in trees (Rudinsky & Vité 1959; Harris 1961; Essiamah & Eschrich 1986; Barnett et al. 1993; De Faÿ et al. 2000), woody shrubs (Hargrave et al. 1994) and herbaceous plants (Tang & Boyer 2002). However, the difficulty in clarifying the functional region of water transport at the cellular level has also been pointed out (Čermák et al. 2002). To prevent the diffusion or flow of dye-carrying water after sampling of the stem, Sano et al. (2005) proposed the dye injection method, which enabled the visualization of the water-conducting xylem tissue of living trees at the cellular level using freeze drying and dye insoluble solution when samples were embedded, and optimal conditions for visualizing water-conducting pathways by this method was determined (Umebayashi et al. 2007). In this study, we examined the patterns of dye ascent of 44 deciduous broadleaved species with ring-porous and diffuse-porous wood structure, using the improved dye injection method. We especially focused on the interspecific variation of the water Downloaded from Brill.com10/05/2021 06:51:38AM via free access 248 IAWA Journal, Vol. 29 (3), 2008 Umebayashi et al. — Conducting pathways in temperate trees 249 transport region between ring-porous and diffuse-porous species and within ring- porous and diffuse-porous species at the cellular level. In addition, we examined the correspondence between tissue regions that remained sap-filled throughout the winter and tissue regions taking up dye. MATERIALS AND METHODS Plant materials Forty-four deciduous broadleaved species (eight ring-porous and 36 diffuse-porous species) growing in the Shiiba Research Forest of Kyushu University (32° 22' N, 131° 09' E; elevation 880–1150 m) in Japan were used in this study (Table 1). According to the temperature and precipitation data from 2003 to 2005 in the Shiiba Research Forest (elevation 600 m), the yearly mean air temperature was 12.7 °C. The daily maximum temperature for 2003, 2004, and 2005 was 33.0, 32.5, and 33.4 °C, respectively, and the minimum temperature for the three years was -9.1, -8.5, and -8.3 °C, respectively. The average yearly mean precipitation for the three years was 2918 mm. Introduction of the dye solution and collection of samples Dye injection experiments were performed from 11:00 to 14:00 on sunny days from mid-July to August in 2003, 2004, and 2005. A watertight collar was set at a stem height of 1.0 m and filled with distilled water. The southern half of the stem was cut with a fine hand saw under water and the water was then replaced with an aqueous 0.2% acid fuchsin solution that had been filtered through a 0.22μ m filter (GV, Millipore, Billerica, USA) before the experiment. To keep water on the cut surface and avoid the adhesion of debris, the stem was wrapped with a fine cotton fabric during the replacement. After perfusion for 30 min, sample discs of 2 cm thickness were taken at 0, 10, 30, and 50 cm above the dye injection height, and at 50 cm intervals up to 1.0 m to 10 cm below the maximum height of the stained xylem. All discs were frozen by liquid nitrogen (LN2) immediately, transferred to the laboratory, and freeze-dried at -50 °C (Umebayashi et al. 2007). Two experiments were carried out for each species. Observation of the dye distribution in the xylem The surface of the sample discs was trimmed with a razor blade and the dye distribution in the cross section of stem xylem was observed macroscopically. Following this, the stained portion of the disc was split into small blocks, the surfaces of the cross section were planed with a sliding microtome, and the dye distribution at the tissue level was observed with a stereomicroscope (SMZ800, Nikon, Tokyo, Japan). Subsequently, the small blocks containing at least one annual ring were trimmed and embedded with an epoxy resin (Sano et al. 2005). Finally, thin sections of 4–7 μm were cut from the embedded blocks with a semi-ultramicrotome (RM2045, Leica, Wetzlar, Germany) and the dye distribution in each xylem cell was observed by light microscopy (Optiphoto-2, Nikon, Tokyo, Japan). Images were obtained using a digital microscope camera system (DP70, Olympus, Tokyo, Japan) attached to the stereomicroscope and to the light microscope. Downloaded from Brill.com10/05/2021 06:51:38AM via free access 250 IAWA Journal, Vol. 29 (3), 2008 Umebayashi et al. — Conducting pathways in temperate trees 251 Table 1. Tree height, diameter at breast height, the maximum dye ascent height and the stained

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