Plant materials and growth conditions
L. bicolor seeds were collected from Dongying, China (118°37’43” E, 37°54’38” N), and stored at 4°C. The seeds were sterilized in 0.5% (w/v) sodium hypochlorite solution for 15 min and then cleaned with sterile-distilled water. The seeds were sown on well-washed river sand in plastic pots (16 cm in diameter; after the leaves emerged, the plants were watered with Hoagland’s nutrient solution), which were placed in a growth chamber with 600 μmol m–2 s–1 light (15-h day/9-h night photoperiod), a temperature of 25 ± 3°C/22 ± 3°C (day/night), and a relative humidity of 60/80% (day/night).
Combined NaCl and melatonin treatment
When the seedlings reached the six-leaf stage, they were subjected to NaCl and melatonin treatments. For the NaCl treatment, the seedlings were treated with Hoagland’s nutrient solution containing NaCl, which was increased by 50 mM every 12 h to a final concentration of 300 mM to avoid salt shock. When the NaCl concentration reached 300 mM, melatonin treatment starts, which will treat for 15 days. The control seedlings were treated with Hoagland’s nutrient solution only. To research the effects of melatonin on salt tolerance in L. bicolor, the NaCl-treated (the NaCl concentration gradually increased to 300 mM) and control seedlings were irrigated with 0 or 5 μM melatonin (based on the pre-test with various concentrations of melatonin, Fig. S1), which dissolves in the above Hoagland’s nutrient solution. The L. bicolor seedlings were treated with various combinations of salt and melatonin every 12 h for 15 consecutive days. Five replicates (3 plants per replicate) were performed for each treatment. After 15 days, the leaves were collected to determine the biological indicators.
Physiological index measurements
The dry weights of seedlings after 15 days of the treatments were measured as described by Yuan et al. [10]
Melatonin quantification
The melatonin content of L. bicolor leaves was quantified as described by Sun et al. [54]. Briefly, 0.3 g L. bicolor leaves were ground into powder in liquid nitrogen, mixed well with 1.5 mL methanol, and incubated overnight at 4℃. The solutions were centrifuged for 10 min at 10,000 ×g at 4℃, after which the supernatant was transferred into new test tubes and the liquid was allowed to evaporate. The remaining residues were dissolved in 0.75 mL methanol. A fluorescence detector system (L3000; Rigol Technologies, Beijing, China) was used to determine the melatonin concentration by the area of the peaks identified during high-performance liquid chromatography.
Ion content measurement
Leaf samples (5 g) were placed into test tubes containing 10 mL ddH2O and the tubes were placed in a boiling water bath for 3 h, after which the samples were filtered through filter paper. The supernatant was made up to a volume of 25 mL with ddH2O. The Na+ and K+ contents were measured using a flame spectrophotometer (Model 2655-00 Digital Flame Analyzer; Cole-Parmer Instrument Company, Vernon Hills, Illinois, USA), and the Cl– content was measured using an ion chromatograph (ICS-1100 ion chromatograph; Thermo Fisher Scientific, Waltham, MA, USA), as described by Lin et al. [55]
Characterization of the L. bicolor salt glands
L. bicolor leaves (Fig. S2) were cleared using Carnoy’s solution (mixed solution of ethanol and acetic acid (3:1, v/v)), as described by Kuwabara and Nagata [56](2016), after which they were fixed onto microscope slides using Hoyer’s solution [57](Yuan et al., 2014). The diameters and densities of the salt glands were determined using a Nikon fluorescence microscope (ECLIPSE 80i; Nikon, Tokyo, Japan) at ×200 and ×100 magnifications with a standard DAPI filter set under UV excitation (330–380 nm). Digital images were taken using a charge-coupled device camera. Fifteen leaves were measured per treatment, with the measurements taken at the same position on each leaf. The salt glands were counted for a given leaf area to calculate the salt gland density on the abaxial surfaces of the leaves [17]. The total number of salt glands is equal to the salt gland density multiplied by the leaf area. The salt secretion levels under different NaCl and melatonin treatments were determined using the leaf disk method, as described by Lu et al. [26].
qRT-PCR analysis
The nucleotide sequences of ion homeostasis (LbHTK1, LbSOS1, LbPMA, and LbNHX1) and vesicle transport (LbVAMP721 (vesicle-associated membrane protein 721), LbVAP27 (syntaxin from plants 27), and LbVAMP121 (vesicle-associated membrane protein 721)) genes in L. bicolor were obtained according to the second-and third-generation RNA sequences [3]. A BLAST search for homologous genes was carried out in both L. bicolor and other species, and homologous sequences were downloaded. Primers were designed for cloning the conserved region sequences (800 bp). The conserved region sequences of LbHTK1, LbSOS1, LbPMA, LbNHX1, LbVAMP721, LbVAP27, and LbVAMP121 were obtained. Beacon Designer (Premier Biosoft, Palo Alto, California, USA) was used to design primers for these seven genes (Table S1). AceQ Universal SYBR Green qPCR Master Mix (Vazyme Biotech, Nanjing, China) and a real-time quantitative PCR instrument (Bio-Rad Laboratories, Hercules, California, USA) were used to perform the real-time PCR. The relative expression of each gene was calculated using the 2–△△Ct method [17, 58], with the housekeeping gene LbTUBULIN used as an internal reference.
Statistical analysis
The statistical analysis was performed using the SPSS software package (version 19.0; IBM, Armonk, New York, USA). The statistical significance was determined using an analysis of variance (ANOVA), and significant differences (P < 0.05) between the values were determined using Duncan’s multiple range test.