Chn analyzer

After 6 weeks of growth in the climate chamber, the light saturated rate of photosynthesis at atmospheric CO₂ concentration per unit leaf area (A_sat) was measured on the middlemost leaf of each plant, using a LICOR 6400 (Portable Photosynthesis analyser, LI-COR Inc. Lincoln, NE, USA) at atmospheric CO₂ concentration (ca. 380 µmol mol⁻¹ in the leaf chamber), at 20°C growth temperature, and a PPFD of 1,500 μmol m⁻² s⁻¹. Before the measurement, leaves were prelighted for 5 min at a high light intensity to initiate photosynthetic induction. To assure that the photosystem is fully saturated, the leaf was prelighted, and the measurement was carried out at a high light intensity. Three measurements were done on the same leaf when steady state was reached, which were averaged and further treated as one data point. The leaf sample used for the photosynthesis measurement was dried in an oven at 60°C for 3 days, and nitrogen (N) concentration was measured with a CHN analyzer (Carlo Erba, Milan, Italy) to calculate the nitrogen content per unit leaf area. In a previous study, the nitrate concentration was analyzed in the leaves of J. vulgaris, and only trace amounts of nitrate were found, indicating that J. vulgaris is not a nitrate accumulator, and we therefore used total nitrogen content to calculate PNUE.