Study site and materials

Our study site consisted of an artificially constructed open-sky empty lot on the campus of Ishikawa Prefectural University, Nonoichi, Ishikawa Prefecture, Japan (36°30′N, 136°35′E). The mean annual temperature and precipitation (2002–2008) at the study site were 14.3°C and 2161 mm, respectively (data from IPU-1 at Ishikawa Prefectural University). The study was conducted in August 2006, during the hottest part of summer. The mean air temperature at the site in August 2006 was 27.6°C. The study species, Oenothera biennis L. (Onagraceae), is a monocarpic perennial herb that was introduced to Japan from North America and it is a pioneer species in open, disturbed habitats, such as roadsides, in Japan45. Three single-stem individual bolting rosettes (i.e., vertical stems, 0.8–1.3 m tall) that were naturally growing at the site were measured in situ. These three plants, hereafter referred to as Plants 1, 2 and 3, were isolated and had no shading from neighbors.

Measurement of photosynthesis

We measured the photosynthetic rates of a total of eight leaves from the three plants on August 3 (Plant 1), August 24 (Plant 2) and September 4 (Plant 3), 2006 using a portable photosynthesis system (LI-6400; LI-COR, Lincoln, USA) equipped with an LED light source (LI-6400-02B). For each leaf, photosynthetic light response curves were repeatedly measured (4–6 times) from dawn to dusk. During all but the first (dawn) measurement for each leaf, the incident PPFD on the leaf surface was lowered progressively (2000, 1500, 1000, 750, 500, 250, 125, 63, 32 and 0 μmol m−2 s−1). Incident PPFD, supplied as natural sunlight to each leaf surface, was determined prior to the dawn measurements using a quantum sensor (IKS-27; KOITO Kogyo, Yokohama, Japan) temporarily placed on the center of each leaf lamina. During the dawn measurements, the highest PPFD supplied by the LED was controlled so that it did not exceed the recorded in situ incident PPFD to avoid artificially stimulating the leaves with excessively high light intensities. During each measurement, the PPFD was kept constant at each decreasing level until equilibration. Leaf conductance to H 2 O (g actual ; mol m−2 s−1), leaf transpiration rate (Tr actual ; mmol H 2 O m−2 s−1), leaf temperature and vapor pressure deficit based on leaf temperature were simultaneously calculated using the LI-6400 system. In the following analyses, leaf conductance was regarded as equivalent to leaf stomatal conductance, assuming that the leaf boundary layer resistance was negligible. The CO 2 concentration of the air entering the leaf chamber was controlled at 350 ppm.

Measurement of incident PPFD

We chose two consecutive clear days with very similar weather to conduct measurements (Fig. 7). For Plant 1, a diurnal course of photosynthetic light response curves was measured on the first day (August 3) as described above and a diurnal course of light intensity on the same leaves was measured on the second day (August 4). Light data from August 4 were used as an estimate for photosynthesis on August 3, assuming that the weather conditions were the same over these two days. For Plants 2 and 3, rainy or cloudy weather prevailed after the photosynthetic measurements, so neither incident light intensity nor the photosynthetic rate under natural sunlight was estimated. We used small, leaf-mounted gallium arsenide photodiodes (150 mg: G1118; Hamamatsu Photonics, Hamamatsu, Japan) to estimate incident PPFD on the leaves as described by Nishimura, et al.46. Photodiodes were calibrated individually against a quantum sensor (IKS-27; Koito, Yokohama, Japan). After measurement of photosynthetic light responses, one photodiode was mounted on the adaxial surface of each leaf by using adherent tape. The photodiodes were connected to a voltage logger (Thermodac-F; Eto Denki, Tokyo, Japan) using light telephone wires. The incident PPFD on each leaf was recorded every 10 min for 24 h.

Figure 7 Diurnal changes in open-sky PPFD on the two measurement days in 2006. Full size image

Data analysis

As described above, the photosynthetic rate under high light intensity was not quantified during the first (dawn) measurement for each leaf. To obtain these data, we fitted a non-rectangular hyperbola47 to each of the PPFD-photosynthesis relationship models to estimate the photosynthetic rate at high PPFD ranges (R2 > 0.999 for all cases). We also fitted an empirical hyperbola9 to each of the PPFD-g actual relationship models (R2 > 0.965 for all cases) and the PPFD-Tr actual relationship models (R2 > 0.981 for all cases) to estimate g actual and Tr actual at high PPFD ranges, respectively. Curve-fitting was conducted with R software, version 3.0.0 (R Foundation for Statistical Computing, Vienna, Austria). Next, the photosynthetic rate, stomatal conductance and transpiration rate were estimated by linear interpolation for any PPFD intensity and values between the two successive measurements for each leaf. Pre-dawn and post-sunset photosynthetic response curves were assumed to be the same as those obtained for the first and final measurements, respectively. The net photosynthetic rate for every 10-min increment was calculated for each leaf from the estimated light response curves and the estimated incident PPFD. The dark respiration rate was estimated as the absolute value of the net photosynthetic rate at PPFD = 0. The gross photosynthetic rate at each moment (P gross_actual ) was calculated as the sum of the net photosynthetic rate and the dark respiration rate at that moment. Instantaneous transpiration rates and stomatal conductance at each moment were calculated for each leaf from the incident PPFD data in the same manner as described for the photosynthetic rate. The sum of daily net photosynthesis (P day_actual ) and transpiration (Tr day_actual ) was calculated by integrating these values.