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.