6400 xt infrared

The relative water content was measured on the 3rd leaf using the protocol in [83 ]. Free proline content was determined according to [84 ] with slight modifications. Homogenized leaves (100 mg) were incubated in 3 ml of 3% sulphosalicylic acid at 96 °C for 10 minutes. Samples were clarified by centrifugation and 1 ml of supernatant was mixed with 2 ml of 50% acetic acid, 2 ml of 2.5% acidic ninhydrin solution and boiled for another 30 min. The reaction product was liquid-liquid extracted by 5 ml of toluene and the absorbance of the toluene fraction was measured at 520 nm. The concentration of proline was determined using a standard curve (0-30 μg) and expressed as μg·mg^− 1 of protein. Estimation of protein concentration was according to [85 (link)] by spectrophotometric measurement of absorbance of PBS (100 mM, pH 7.8) buffered leaf extracts at 260 and 280 nm. The level of lipid peroxidation was determined by measuring the MDA concentration as described in [86 (link)]. Gas exchange measurements were made on the 2nd leaf using the LI-COR 6400-XT infrared gas analyzer with attached leaf chamber LI6400-40 (Li-COR, Biosciences). The measurements were performed with a CO₂ reference concentration of 400 μmol·mol^− 1, an air flow of 200 μmol·s^− 1, block temperature of 20 °C, photosynthetic photon flux density of 1800 μmols·m^− 2·s^− 1 and relative humidity between 55 and 65%.

We measured A_max, g and E parameters using a LI-COR 6400XT infrared gas analyser (IRGA; LI-COR Biosciences, Lincoln, NE, USA). All measurements were conducted using six seedlings of each species under each treatment from 11:00 to 14:00 h on August 17, 2014, which was a sunny day. Light was supplied by an LED lamp (LI-6400-02B, LI-COR Biosciences), and the light levels were kept at 1500 μmol·m⁻² s⁻¹, which is above the light saturation point for all four species according to the light response curves before the measurements. The external CO₂ was provided by portable tanks containing a CO₂/air mixture with a concentration of 400 μmol·mol⁻¹. A CO₂ injector (LI-6400-01, LI-COR Biosciences) was used to control the output of the tanks. The temperature was maintained at 25–28 °C, and the relative humidity was kept at 25–30%. Then, we divided A_max by E to calculate the instantaneous water use efficiency (WUE_i).
We examined the chlorophyll fluorescence parameters of leaves that had been kept under dark conditions for 30 min in the morning between 05:00 and 06:00 h on August 17, 2014. We also measured the maximum quantum yield of photosystem II (PSII, F_v/F_m) with the formula (F_m − F_o)/F_m, using an LI-6400-40 leaf chamber fluorimeter (LI-COR Biosciences).