Effective use of high CO2 efflux at the soil surface in a tropical understory plant

Many terrestrial plants are C3 plants that evolved in the Mesozoic Era when atmospheric CO2 concentrations ([CO2]) were high. Given current conditions, C3 plants can no longer benefit from high ambient [CO2]. Kaempferia marginata Carey is a unique understory ginger plant in the tropical dry forests of Thailand. The plant has two large flat leaves that spread on the soil surface. We found a large difference in [CO2] between the partly closed space between the soil surface and the leaves (638 µmol mol−1) and the atmosphere at 20 cm above ground level (412 µmol mol−1). This finding indicates that the plants capture CO2 efflux from the soil. Almost all of the stomata are located on the abaxial leaf surface. When ambient air [CO2] was experimentally increased from 400 to 600 μmol mol−1, net photosynthetic rates increased by 45 to 48% under near light-saturated conditions. No significant increase was observed under low light conditions. These data demonstrate that the unique leaf structure enhances carbon gain by trapping soil CO2 efflux at stomatal sites under relatively high light conditions, suggesting that ambient air [CO2] can serve as an important selective agent for terrestrial C3 plants.

November to March (Ishida et al. 2006). The soil is of sandstone origin and acidic (around 4.5 in pH), and has relatively poor nutrients with high porosity. Hopea ferrea Lanessan (Dipterocarpaceae) is the predominant tree with tall canopies (approximately 25-35 m high) in the evergreen forest. Details in landform and soil characteristics are shown in Pitman (1996) and Murata et al. (2009).
The examined ginger plant (Kaempferia marginata Carey, Zingiberaceae) is a drought-deciduous perennial herb. The plants are usually found near the roadside in the dry evergreen forests with dense canopies, and inside the drought deciduous forests with sparse canopies in the Sakaerat Environmental Research Station. Thus, the ginger plant seems to favor to a relatively light understory.

Measurements of leaf size distribution
We selected a population of the ginger plant found roadside in the dry evergreen forest.
To examine the ontogenetic variations of leaf form, we measured the leaf area, leaf The daily total PPF at the study site relative to that of the open site was 6.4%. The understory light levels in tropical evergreen forest are approximately 1% of full sunlight or sometimes less than 1% (e.g., Ashton 1992). Because the ginger plants are not found in deeper-shaded sites in the evergreen forests, the light levels of approximately 6.4% relative to full sunlight appear to be required to maintain a population of the ginger plant.

More detailed methods in the photosynthetic capacity measurements
The leaf photosynthetic capacity in eight individuals was measured with an open, portable measurement system (LI-6400, LI-COR, Lincoln, NE). The measurement was conducted in six healthy, mature leaves. The leaf chamber with 6 cm 2 was used and the red-blue RED lamp unit was utilized as a light source. Photosynthetic light-response curves were measured on the same eight leaf blades under 600 and 400 µmol mol -1 CO 2 in the inlet gas stream with LI-6400. The values of 400 and 600 µmol mol -1 CO 2 were approximate and corresponded to daily mean air CO 2 concentrations at the 20 cm above and just below the leaf blades, respectively. PPFs were decreased stepwise from 800, 500,200,100,70,40,30,20,10,7,3, to 0 µmol m -2 s -1 . Light compensation points and apparent quantum use efficiencies were calculated from linear regressions under very low PPFs (from 0 to 10 µmol m -2 s -1 ). The mean leaf temperature during these measurements was 29.2°C which approximately corresponds to the daytime leaf temperature. Photosynthetic ambient air CO 2 -response curves were measured on the 6 same seven leaf blades under 500 and 40 µmol m -2 s -1 PPF with LI-6400. In the values that were exposed to sun-flecks and in those without sunflecks during the daytime, the values of 500 and 40 µmol m -2 s -1 PPF were used, respectively (see Supplementary Fig.   2). The [CO 2 ] in the inlet gas stream with LI-6400 increased stepwise from 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, to 1000 µmol mol -1 . Effeltrich, Germany), according to Bilger et al. (1995). The fiber-optic cable was connected with the clear top-cover of the LI-6400 chamber, while the angle (60°) and the distance between the leaf surface and the fiber-optic cable were manually adjusted.

Maximum fluorescence yield (F m ) and dark fluorescence yield (F o ) in photosystem II
(PSII) were determined just before dawn. Just after the measurement of leaf gas exchange, we supplied a saturated-light pulse to the leaf surface. Maximum fluorescence (F m ') and steady-state fluorescence (F) in the light-adapted state of PSII were measured during the daytime. Chlorophyll fluorescence parameters were calculated, according to Genty et al. (1989). The potential maximum quantum yield of PSII (F v /F m = (F m -F o )/F m ) was calculated from the dark-time measurements made before dawn. For each daytime measurement, the effective quantum yield of PSII (Φ PSII = (F m '-F)/F m ') was calculated. Assuming that photosystem I and II absorb equal amounts of light and the leaf absorbance of lamina is 0.84, the electron transport rate through PSII (ETR) was calculated as, ETR = 0.5 Φ PSII 0.84 PPF (at the leaf surface).

Measurements of the nitrogen and stable carbon isotope ratio in lamina and the number and size of stomata
After all measurements, we collected the leaves and then cut leaf discs with a borer. The leaf discs were oven dried (70°C, 72 hr) and weighed to determine leaf dry mass per unit leaf area (LMA). The total nitrogen (N) and carbon (C) contents within the leaf discs were measured with an N-C analyzer (Sumigraph NC-900, Sumitomo-Kagaku, Osaka).
To estimate the averaged internal CO 2 concentrations in leaves for a long time, the stable carbon isotope ratios (δ 13 C) in lamina were determined with an isotope ratio mass spectrometer (DELTA V Plus, Thermo Fisher Scientific Inc., Cambridge, UK).
The δ 13 C values were expressed in delta notation relative to a PD Belemnite standard: δ 13 C = (R sample -R standard -1) 1000 (‰), where R sample is the 13 C/ 12 C ratios of the samples and R standard is the 13 C/ 12 C ratio of the standard.

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The numbers and the pore length of stomata in the adaxial and abaxial leaf surfaces were determined by obtaining replicas of the surface of four healthy leaves with a celluloid plate (Universal Micro-printing, SUMP, Tokyo, Japan).  Appl. Ecol. 33, 1366-1378(1996.
Sakurai, K., Tanaka, S., Ishizuka, S., Kanzaki, M. Differences in soil properties of dry evergreen and dry deciduous forests in the Sakaerat Environmental Research Station.