Gas exchange and fluorescence measurements

Leaf gas exchange and fluorescence parameters of the central leaf section were simultaneously measured using a LI-6400-40 leaf chamber fluorometer (Li-Cor, Inc., Nebraska, USA) equipped with a 2 cm² cuvette. One branch for the ABA and control treatments was taken from each of four plants, with the youngest fully expanded leaf from four branches measured for each treatment. The measurements were made at a saturating photon flux density (PPFD) of 1600 μmol m^-2s^-1 measured using the internal quantum sensor within the leaf chamber and leaf temperature of 25°C for all four species. Leaves were exposed to a contaminant and pollutant free flux of synthetic air, composed of a mixture of nitrogen (80%), O₂ (20%) and CO₂ (385 ppm). The relative humidity of the air flow (500 μmol s-1) was maintained at 40–50%. To reduce diffusion leaks through the chamber gasket [37], a supplementary external chamber gasket composed of the same polymer foam was added to create an interspace between the two gaskets (i.e. a double-gasket design with a 5 mm space separating the internal and external gaskets). Then the CO₂ and H₂O gradients between the in-chamber air and pre-chamber air were minimized by feeding the IRGA exhaust air into the interspace between the chamber and the pre-chamber gaskets [38].

The variable J method has proven to be effective in determining g_m in both well-watered and drought stressed rice varieties [2], we therefore chose this approach to assess the effect of ABA on g_m in the four plant species. Mesophyll conductance was calculated using the variable J method involving simultaneous measurements of gas-exchange and chlorophyll fluorescence parameters as described by Harley et al. (30) and Loreto et al. (31) (Eqs 1 and 2):

where the electron transport rate (J_F) is calculated from fluorescence [39]:

where F_m is the fluorescence maximum and the partitioning factor (β) between photosystems I and II was considered to be 0.5 and leaf absorbance (α) (0.85) [40].

The CO₂ compensation point to photorespiration (Γ*) was measured on individual attached leaves of intact plants by increasing C_i at four different levels of photosynthetically active radiation [41, 42]. Levels of respiration in the light (R_d) were analysed using the Kok method [43, 44]; and respiration in the dark (R_n) was measured by switching off the light in the cuvette, when CO₂ release from the leaf had become stable for approximately five to ten minutes this was recorded and considered to represent R_n [45]. Values of Γ*, R_d and R_n used in the calculation of g_m utilising the variable J method are given in Table 1. Total conductance to CO₂ (g_tot) was calculated as:

± indicates one standard deviation.