Quantitative real-time PCR
Total RNA was extracted by Trizol reagent, and one μg RNA was used for inverse transcription to synthesize the first strand of cDNA following the manufacture’s instruction (Takara RR047 kit, Dalian, China). The cDNA was used to test gene expression level using Starlighter SYBR Green qpcr Mix (Beijing Qihengxing Biotechnology Co., LTD, FS-Q1002 kit) with gene specific primers (Table S2) with an qTOWER3G (analytik jena). Amplification of alfalfa β-actin gene (JQ028730.1) was used as an internal control to normalize the gene expression. The 2-∆∆CT measurement method was used to calculate the relative expression level (Liu et al., 2019). The data of the relative expression levels were means derived from three biological replicates.
Stomatal conductance and stomatal movement assay
Leaves of the second internode from the top of four-month-old plants were used for analyzing the stomatal movement. Firstly, the adaxial surface of leaf was immersed in a buffer (50 mM KCl, 10 mM MES, and 0.1 mM CaCl2, pH 6.15) under a cold light source (200 μmol m-2 s-1) for 4h. Then, the leaf was transferred to a new buffer with additional 0 μM ABA (CK), 25μM ABA, or 15% PEG6000. The leaf was fixed in the FAA solution after treatment for 40 min. The photograph of the stoma on the abaxial surface of leaves was captured under an optical microscope. The size of the stomatal aperture was measured using Image J software. At least 80 stomata were measured from three biological repeats (each one contained three leaves), that were used for statistical analysis.
Stomatal conductance was measured with a LI-COR/LI-6400 portable photosynthesis measurement system with an LED light source at 200 μmol m-2 s-1, airflow was set to 500 μm and the CO2 concentration at 800 ppm (Pan et al., 2020). Eight leaves were treated as one biological replicate, and three biological replicates were given in the tests.