Winrhizo program

After 3 days of Na₂CO₃ treatment, the survival rate of the rice seedlings was determined. Ten rice seedlings were randomly selected from the 25 seedlings in each treatment group and scanned using an Epson Expression 10000XL (Epson America Inc., Long Beach, CA, United States). The resulting images were digitized using the WinRHIZO program, according to the manufacturer’s instructions (Regent Instruments Canada Inc., Ville de Québec, QC, Canada), and the average shoot length (SL, the length of the longest leaf), total root length (TRL), total root surface area (RSA), root diameter (RD), total root volume (RV), and root number (RN) were determined. After scanning, the seedlings were cut and divided into shoots and roots, and their fresh weights measured. These samples were then dried in a forced-air-driven oven at 105°C for 2 h, followed by 70°C, until a stable mass was reached, prior to the determination of dry mass. The roots were rinsed four times with deionized water and then dried. Individual seedlings were classified as dead if all the leaves were dry and brown.

A total of 5 plants of alfalfa seedlings for each replication was randomly selected from the 25 seedlings with 5 replications in each treatment after 48 h of alkaline stress. The roots of each plant seedling were scanned using an Epson Expression 10000XL (Epson America Inc., Long Beach, CA, United States). The resulting images were digitized using the WinRHIZO program according to the instructions of the manufacturer (Regent Instruments Canada Inc., Ville de Québec, QC, Canada), and the root parameters, total root length, total root surface area, total root volume, average root diameter, and root number of forks were determined. After scanning, the seedlings were cut and divided into aboveground parts and roots. The fresh weight (FW) of alfalfa seedlings was estimated after washing with deionized water and blotting on paper towel. These samples were then dried in a forced-air-driven oven at 105°C for 2 h, followed by 70°C until maintaining a stable mass prior to the determination of dry mass (Zhang et al., 2017 (link)).

Throughout the experiment, the height, leader shoot, and stem diameter of the Scots pine trees were monitored monthly (as detailed in Methods S4). On a seasonal basis, leaf gas exchange (light-saturated photosynthesis (A_net) and stomatal conductance (g_s)) were measured using a LiCor 6400 system (LI-COR Biosciences, Lincoln, NE, USA). Specifically, 25 south exposed needles were enclosed in the 2 × 3 cm chamber, and A_net and g_s were measured under 400 μmol mol⁻¹ CO₂, 1000 PAR, local humidity and temperature, and a stomatal ratio of 1. At the end of the first growing season of the trees in the mesocosms (on August 28^th 2020), predawn leaf water potential (Ψ) was measured on current-year twigs between 04:00 and 05:30 a.m., using a Scholander-type pressure chamber (PMS Instrument Company, Albany, NY, USA) in steps of 0.05 MPa. The leaf area of the needles was also determined at the end of the first growing season of the trees in the mesocosms by randomly collecting 30—40 needles throughout the whole crown of the trees and scanning them with a flatbed scanner (EPSON Expression 11000XL, EPSON, Suwa, Nagano, Japan). The scanned images were analyzed with the WinRHIZO program (version 2013, Regent Instruments Inc., Chemin Sainte-Foy, Quebec, Canada) to determine the leaf area of the needles (Albaugh et al. 2020 (link)), which was normalized per needle.