Li 6400

The plants were monitored daily for root necrosis, growth of visible mycelia on the root surface, wilting of leaves and mortality.
Gas exchange measurements were conducted at 0, 2, 4,6 and 8 dpi using a CO₂/H₂O diffusion porometer equipped with a broad-leaf LED cuvette (LI-6400, LI-COR, Lincoln, Nebraska, USA). All the measurements were conducted under steady-state conditions of 23°C (leaf temperature), between 50% and 60% relative humidity and 400 ppm CO₂ concentration (in the reference air), 250 µmol m⁻² s⁻¹ PPFD, and 500 mL min⁻¹ air flow.
To analyze the activity of the enzyme RuBisCO, an A/C_i-Curve was performed at 8 dpi with the LI-COR LI6400 using the default program of the device. Leaf CO₂ uptake (A) versus intercellular CO₂ concentration (C_i) curves provide information about the limitation of photosynthesis.
The slope of an A/C_i-curve was used to calculate RuBisCO activity (V_cmax), the maximum rate of electron transport (J_max) and mitochondrial respiration (R_d). The data were analyzed with a Microsoft Excel based macro (http://landflux.org/Tools.php) on the models of [33] –[35] .

For light-saturated photosynthesis, the middle parts of fully expanded leaves were measured using a portable open gas exchange system (Li-6400, Li-COR Inc., Lincoln, NE, USA) with a 2 × 3 cm leaf chamber (Li-6400-02 LED, Li-COR Inc., Lincoln, NE, USA) at an ambient CO₂ concentration, similar to Cui et al. (2016 (link)), but with light intensity set at 1,500 μmol m⁻² s⁻¹, leaf temperature set at 25°C. For measurements of diurnal variation of the net photosynthesis, measurements were taken on clear days around flowering stage with natural fluctuation of air temperature and vapor pressure deficit. The PPFD used for each assessment was provided by the artificial light source (Li-6400-02 LED, Li-COR Inc., Lincoln, NE, USA), which was set to provide the same PPFD as solar intensity that was tracked by an external light sensor of Li-6400 near leaf chamber.

The height and basal diameter of the walnut saplings were measured on 18 October 2016. Basal diameter was measured at 3 cm above the ground with a vernier caliper. To determine a tree’s crown diameter, we measured its crown extensions north, south, east and west with a tape.
A portable photosynthesis system (LI-6400; LI-COR, Inc., Lincoln, USA), with a red/blue light-emitting diode (LED) light source (LI6400-02B) mounted on a 6-cm² clamp-on leaf chamber, was used to determine the photosynthesis rate under sunny and windless weather conditions. The net CO₂ assimilation rate (Pn) and transpiration rate were measured on fully expanded walnut sapling leaves at similar development stages with a portable open-flow gas exchange system (LI-6400, LI-COR Inc., USA) during the late morning (9:00–11:00 h), with three duplications per treatment. Pn was measured one time along the season on 19 October 2016. In all cases, the air relative humidity, CO₂ concentration and photon flux density were maintained at 60%–70%, 380 mmol·mol⁻¹ and 800 mmol·m⁻²·s⁻¹, respectively.

The youngest fully expanded leaves (diagnostic leaf) were sampled at 29, 51, and 88 days of treatment (DOT) and separated to blade and petiole for mineral analysis. Photosynthesis (A_n, LI-6400, Li-Cor Inc., Lincoln, NE, USA) and stomatal conductance (g_s, LI-6400) of matured, sun-exposed leaves were measured after 91 days of growth in three plants per treatment between 10:00 and 13:00. The reference CO₂ in the LI-6400 was set to 400 ppm, light to 1,000 µmol m^-2 s^-1, and temperature and humidity levels were ambient (∼30°C and 40% RH in the greenhouse). All the plants were harvested after 190 days of growth, divided into tuberous roots, stem, and leaves. The total fresh weight was recorded and all the samples were oven-dried at 70°C for 48 h, and they were then re-weighed to ascertain their water content and dry mass (DM). Finally, leaves of 18 plants (3 replicates per treatment) were stored for subsequent lab carbohydrate analysis.

The second functional leaf of the main stem (from the top of the plant) was used to measure the chlorophyll and gas exchange parameters. A SPAD-502 chlorophyll meter was used to measure the SPAD values of the upper, middle, and lower parts of the leaf, with the average value used as the final SPAD value. Photosynthetic parameters, including net photosynthetic rate (Pn), stomatal conductance (Gs), intracellular CO₂ concentrations (Ci), and transpiration rate (Tr), were determined using the LI-6400 portable photosynthesis system (LI-6400, LI-COR, USA) between 9:00-11:30 A.M. The apparent mesophyll conductance (AMC) was estimated using the rate of Pn/Ci (Liu et al., 2012 ). The conditions in the leaf chamber were: photosynthetically active radiation (PAR) of 1,000 µmol m⁻² s⁻¹, CO₂ concentration of 400 µmol mol⁻¹, and leaf temperature of 26.0 °C. The data were automatically collected every three minutes.

Photosynthesis was measured following the method previously used [55 (link)]. An Arabidopsis leaf was placed inside a controlled-environment chamber using a Li-6400 portable gas-exchange system (Li-6400, Li-Cor, Inc., Lincoln, NE, USA) with saturating light (1200 μmol·m⁻² s⁻¹) and 400 μmol·mol⁻¹ CO₂. The temperature was set to 28 °C.

The contents of chlorophyll were measured according to the method of Lichtenthaler with little modifications [60 ]. Eighty percent (v/v) acetone was used to extract chlorophyll, and the content was estimated according to the absorbances at 470, 646 and 663 nm.
The net photosynthetic rate (Pn) of fully expanded leaves, as well as transpiration (Tr), stomatal conductance (Gs), and internal CO₂ concentration (Ci), were determined by using a portable photosynthesis system (Li-6400, Li-Cor, Lincoln, NE, USA). Those measurements were carried out in the morning from 9:00 to 11:30. The recovery coefficient of Pn was calculated according to the following equation: Pn^NaCl/Pn^CK. Here, Pn^NaCl and Pn^CK stand for the leaf photosynthetic rate measured in the NaCl and CK treatments.
We applied a fluorometer for measuring chlorophyll fluorescence (Li-6400, Li-Cor, Lincoln, NE, USA). Fv/Fm was calculated by using (F_m − F₀)/F_m, where minimum (dark) fluorescence (F₀) was obtained by applying to measure light pulses at low frequency (0.03 μmol m⁻² s⁻¹ for 1 s). The maximum fluorescence (F_m) was determined by applying a saturating light pulse (6000 μmol m⁻² s⁻¹ for 0.8 s) to a dark-adapted sample [61 (link)].