Seedlings of each cultivar were then

exposed to different N deficiency stress treatments at the five-leaf stage. Hoagland’s solution without N [Ca(NO3)2·4H2O] was then added to maintain various N deficiency treatments [20], including mild stress [N2: 1.5 mmol L− 1 Ca(NO3)2·4H2O], moderate stress (N1: 0.15 mmol L− 1), extreme stress (N0: 0 mmol L− 1) EPZ015666 price and a stress-free control (full strength Hoagland’s nutrient solution, modified). The solutions were refreshed twice a week and the pH of the nutrient solutions was adjusted to 5.5–6.5 every 2 days. An air pump was used for ventilation 24 h per day. Agronomic and physiological traits were evaluated 60 days after treatment. Sixty days after treatments, the tiller number, height (from the pot surface to the end of the longest leaf on the tallest tiller), aboveground biomass, leaf area, and root area were measured. Aboveground biomass was cut at the pot surface and separated into shoots and leaves, the leaf area was determined Roscovitine datasheet with a LI-COR 3100 leaf area meter (Li-Cor, Lincoln, NE) and the root surface area was determined with a root scanner (Epson Expression 1000XL, Japan). Roots and rhizomes were washed free of growth media and all plant samples were treated at 105 °C for 30 min

for fixation and then oven dried at 65 °C until a constant weight was reached. The presence of rhizomes was recorded and the root to shoot weight ratio (R:S) was calculated. Gas exchange measurements were performed two weeks after treatment initiation using a portable open gas exchange system (LI-6400, LI-COR) calibrated to deliver a photosynthetic Liothyronine Sodium photon flux density of 2000 μmol m− 2 s− 1 and an ambient CO2 of 400 μmol mol− 1 (supplied by a LI-COR CO2 injector) and a leaf temperature of (30 ± 1) °C. Data were collected for 2 min at 5-s intervals for three randomly chosen plants from each treatment listed above (eight replications per treatment) on the youngest fully expanded leaf on the longest tiller, as described by Barney et

al. [12]. Net CO2 assimilation (A), transpiration (E), and stomatal conductance (gs) were recorded, and photosynthetic water use efficiency (WUE) was calculated (WUE = net photosynthesis/transpiration). Chlorophyll a and chlorophyll b were extracted with 80% acetone from the same leaf as used for gas exchange measurements. Absorbance was measured at 663 nm and 645 nm for chlorophyll a and chlorophyll b, respectively, using a UV spectrophotometer (UV-2550, Shimadzu). Total chlorophyll content was calculated according to the procedure described by Lichtenthaler and Wellburn [21]. To avoid the negative influence of different cultivars on the evaluation of tolerance, the Low-N tolerance index (LNT) was calculated. This is the ratio of the index under treatment to that of the control (LNT = (value of tested traits under treatments/value of same tested traits under control) × 100%).