13C Analysis and Intrinsic Water Use Efficiency.

Carbon isotope composition (δ¹³C) of whole-shoot biomass and extracted cellulose (46) was analyzed using an elemental analyzer (NA 1110; Carlo Erba Instruments) interfaced (ConFlo II, Finnigan MAT) with a continuous-flow isotope ratio mass spectrometer (Delta Plus; Finnigan MAT), EA-CF-IRMS. Each sample was measured against standard CO₂ calibrated with an isotope standard (International Atomic Energy Agency CH6, accuracy of calibration ± 0.06‰ SD). The precision of sample repeats was 0.08‰ (SD) for bulk biomass samples and 0.13‰ for cellulose and included deviations by subsampling, cellulose extraction, and EA-CF-IRMS measurement. ¹³C discrimination (Δ¹³C, in ‰) was calculated as Δ¹³C = (δ¹³C_air − δ¹³C_plant)/(1 + δ¹³C_plant/1,000) (4). Intrinsic WUE has been defined as: iWUE = A/g_s = 0.625 C_a (1 − C_i/C_a), where A is the net CO₂ assimilation rate and g_s the stomatal conductance (5). The factor 0.625 gives the ratio of the diffusivities of CO₂ and H₂O in air, and C_a and C_i designate the CO₂ concentrations in ambient air and intercellular space. In the growth cabinet, C_a was 422 ± 12 μmol⋅mol⁻¹ with an approximated δ¹³C_air value of −9.7‰. The “simplified” Farquhar model based on nonlimiting mesophyll conductance was applied to estimate C_i/C_a: Δ¹³C = a + (b − a) C_i/C_a. The term a (4.4‰) denotes the fractionation of ¹³CO₂ relative to ¹²CO₂ during diffusion through the stomatal pores, and b (27.6‰), the net fractionation during carboxylation reactions.