Study site and plant material
Studies were performed on a ridge (36°4′N, 138°20′E, 2,226 m above sea level) of the upper subalpine region of the northern Yatsugatake Mountains in central Japan during the winter of 2006–2007. This site experiences strong westerly winds, especially during the winter months. The ground surface remains covered with snow from early December to late May with a maximum depth of 1 m in mid-winter.
The tree forms of the subalpine fir, A. veitchii, vary according to wind exposure, which is influenced by the microtopography of the region. The winter water relations of flagged crown trees [7 cm diameter breast height (DBH), 4 m high] at the most wind-exposed site (W+) were compared with those of normal crown trees (18 cm DBH, 7 m high) at the wind-protected site (W−); the W+ and W− sites were separated by a distance of 300 m (Fig. 1a,b). The needle longevity of the windward shoots was 3 years for W+ trees and 9 years for W− trees. In the period from late winter to early spring, shoot dieback was observed in trees at the W+ site (Fig. 1c), but not in the trees at the W− site. These observations suggest that shoot dieback, which occurs each winter, may be responsible for the shorter longevity of needles on the trees at the W+ site and, consequently, for the formation of flagged trees.
Tree forms of Abies veitchii at the study site (2,226 m above sea level) on the northern Yatsugatake Mountains in central Japan. (a) Flagged crown trees at the wind-exposed site (W+) of a pass. (b) Normal trees at the wind-protected site (W−). (c) Brown coloured needles of A. veitchii trees at the W+ site in late March. These needles were shed by late June.
Temperature measurements
Air temperature was measured every 30 min at a height of 2 m using a Pt-100 sensor (HMP155, Vaisala Corp., Helsinki, Finland) at the W+ site. The temperatures of the xylem (Tx) of A. veitchii (wind-exposed and sun-lit branches and stems at a height of 1.5–2 m) and soil (T0, at the soil surface and T20, at a depth of 20 cm) were measured every 30 min using 0.3 mm thermocouples and recorded with a datalogger (Model CR10, Campbell Scientific Corp., Utah, USA). To measure the xylem temperature, thermocouples were inserted to a depth of 0.8 cm into the xylem of branches (2 cm diameter on a W+ tree and 5 cm diameter on a W− tree) and to a depth of 1.5 cm into the xylem of stems (7 cm diameter on a W+ tree and 18 cm diameter on a W− tree).
Sampling
To monitor water relations over the winter season, westward and sun-lit branches were harvested from each of the four A. veitchii trees at both W+ and W− sites. The trees were different from those used for temperature measurements. Samples were collected just before sunrise only in October, May and June; whereas they were collected at noon during the winter (December–April). In the winter sample collection for pre-dawn water status observations was not necessary as stomata remained closed. The samples were immediately sealed and placed in a plastic bag kept below 5 °C and transported to the laboratory.
Cuticular resistance, relative water content and needle viability
The cuticular resistance to water loss (rc), the relative water content (RWC), and viability of needles were measured as previously described by Hadley and Smith4,5 with slight modifications. Shoots (n = 6) were rehydrated by placing the cut end in water at room temperature (17 °C ± 2 °C) for 24 h. The shoots were then cut into the 1-, 2-, and 3-year-old portions and the cut end was sealed with paraffin. Under dark conditions, the rate of water vapour loss from needles was gravimetrically determined at regular intervals of 1 h until the rate became constant7. Cuticular resistance rc (s−1m) was calculated using an analogy of Ohm’s Law for electrical circuits as follows:
$${I}_{v}=({e}_{s}-{e}_{a})/{r}_{c}$$
(1)
where Jv (g m−2 s−1) is the rate of water vapour loss from needles, es (g m−3) is the water vapour concentration inside a needle (assuming saturation at needle temperature), and ea is the water vapour concentration in air. The measured rc is certainly not the needle resistance but the cuticular resistance because stomata will be closed under dark conditions.
The needle RWC (%) of 2-year-old segments (n = 4) was determined from the measurements of fresh (FW) and dry weight (DW) as follows:
$${rm{RWC}}=({rm{FW}}-DW)/(TW-DW)times 100$$
(2)
where TW is the turgid needle weight after soaking the needles in distilled water for 3 days at 5 °C. The DW of needles was measured after drying the needles at 80 °C for 48 h.
To measure needle viability, needles were collected from 3-year-old shoots (n = 20), and the percentages of needles with >50% green surface area were calculated.
Water potential (ψ)
The water potential (ψ) was determined for approximately 10 cm long tips of 5-year-old shoots (n = 3–5) using a pressure chamber (Model 3000, Soil moisture Equipment Corp., Santa Barbara, CA, USA).
Xylem conductivity
Xylem hydraulic conductivity was measured with modified apparatus as described previously28. For xylem conductivity measurements, 12 cm-long (5-year-old) segments (n = 4) were cut from branches under water in the laboratory. While under water, approximately 5-mm wide bark ends were removed. Subsequently, the segments were fitted to the conductivity system connected to a reservoir with distilled and filtered (0.22 μm) water and the initial hydraulic conductance was measured at a pressure difference of 0.01 MPa. The rate of embolism, described as percent loss of conductivity (PLC), was calculated from the ratio of initial (K) to maximal conductivity (Kmax) of xylem after 10 min flushing (0.5 MPa), according to the following equation:
$$PLC=(1-K/{K}_{max})times 100$$
(3)
Vulnerability analysis
Vulnerability curves were obtained using the air injection method as described previously29. Samples were obtained in December, in addition to the samples obtained monthly. Branches (5-year-old, 12 cm in length, and 8–15 mm in diameter, n = 4) were cut under water and placed in a double-ended pressure chamber with both ends protruding. One end of each segment was attached to the conductivity system. Initial xylem hydraulic conductance was measured as described for the xylem conductivity measurements above. Subsequently, the air pressure was progressively increased until the hydraulic conductivity readings were 0. A vulnerability curve was constructed for each segment by plotting PLC values against the negative air injection pressure. A Weibull curve30 were fit using a nonlinear procedure (SAS Institute Inc.) and a 50 percent loss of conductivity (P50) was estimated.
Safranin staining of stem
To identify functional xylem areas, fresh 2-year-old stem segments (n = 3) were cut under water and connected to a reservoir with 0.1% (w/v) aqueous safranin under a pressure of 0.01 MPa.
Dendrometer measurements
Variations in the diameter of branches and stems were continuously measured using point dendrometers (Type DD, Type DR, Ecomatik Corp., Munich, Germany). Three trees were selected at each site (W+ and W−) for dendrometer measurements. Each branch and stem of these trees was equipped, on their west side, with a point dendrometer at heights of 1.5 m and 2 m at the W+ and W− sites, respectively. Sensing rods of the dendrometers were held with a constant force against the smoothed bark surface of the branches (diameters of 2.2 cm and 5.0 cm at the W+ and W− sites, respectively) and the stems (diameters of 7 cm and 18 cm at the W+ and W− sites, respectively). The resolution of the dendrometers was 1.5 μm and the temperature coefficient of the sensor was less than 0.2 μm. According to the manufacturer, these dendrometers are functional at temperatures as low as −30 °C. Data were collected at 30-min intervals using a CR10X datalogger.
Statistical analysis
The statistical significance of the differences in various measurements between the W+ and W− sites was determined using the Student’s t-test at the 5% probability level. One-way analysis of variance was used to detect significant differences among different shoot ages and between the W+ and W− sites.
Source: Ecology - nature.com
