Plant material and treatment
Glycine soja is a halophytic soybean native to coastal saline soil in Yellow River Delta, China. The seeds of Glycine soja were carefully collected from wild plants, identified by Prof. Hualing Xu and deposited in Dongying academy of agricultural sciences. Experimental research on plants including collection of plant material in this study did not violate any guideline or local legislation. Special permissions and ethical approval were not required to collect and use this wild soybean. Glycine max is a widely planted soybean cultivar in China, and the seeds of Glycine max were obtained from Shandong academy of agricultural sciences. The seeds of Glycine soja and Glycine max are shown in Additional file 1: Figure S1, and the seed specimens were, respectively, conserved in Dongying academy of agricultural sciences and Shandong academy of agricultural sciences without voucher number.
The protocols for seed germination and seedling culture have been reported in our previous study [14]. In July 7, 2017, the seeds of Glycine max were fully soaked in distilled water for 8 h, while the seeds of Glycine soja were soaked in concentrated sulfuric acid for 10 min to remove the hard shell over the seeds. Then, the seeds were placed in petri dishes in the dark between two sheets of filter paper at 25 °C to germinate, and the filter paper was kept wet by spraying Hoagland nutrient solution (pH 5.7). Thereafter, the seedlings were transferred to plastic pots filled with vermiculite, watered with Hoagland solution (pH 5.7) and grown in artificial climatic chambers (Huier, China). The photon flux density, day/night temperature and humidity were controlled at 200 mmol m-2 s-1 (12 h per day from 07:00 to 19:00), 25/18 oC and 65% in the chamber. One month later, uniform plants were selected for salt treatment. NaCl was added to Hoagland nutrient solution (pH, 5.7) incrementally by 50 mM step every day to provide final concentration of 300 mM, and thereafter, salt stress (300 mM NaCl) persisted for 9 days. The newest fully expanded leaves were sampled for measuring physiological and biochemical parameters. After 9 days of salt stress, NaCl in the culture medium were leached completely with Hoagland nutrient solution for examining the recovery of photosystem performance.
Measurements of gas exchange and modulated chlorophyll fluorescence
Gas exchange and modulated chlorophyll fluorescence parameters were simultaneously detected by using an open photosynthetic system (LI-6400XTR, Li-Cor, Lincoln, NE, USA) equipped with a fluorescence leaf chamber (6400-40 LCF, Li-Cor). The temperature and CO2 concentration were respectively set at 25 oC and 400 μmol mol-1 in the leaf cuvette. Pn and gs were simultaneously recorded. Steady-state fluorescence yield was also recorded, and then a saturating actinic light pulse of 8000 μmol m-2 s-1 for 0.7 s was used to produce maximum fluorescence yield by temporarily inhibiting PSII photochemistry for measuring actual photochemical efficiency of PSII (ΦPSII). ETR was calculated as ΦPSII×PPFD×0.84×0.5 [55], and photochemical quenching coefficient was noted for calculating 1-qP.
Measurements of prompt chlorophyll fluorescence, delayed chlorophyll fluorescence and modulated 820 nm reflection transients
The measurements were conducted by using a multifunctional plant efficiency analyzer (MPEA, Hansatech, UK). The leaves were adapted in dark for 30 min, and thereafter, the leaves were illuminated with 1 s red light (627 nm, 5000 µmol photons m-2 s-1) and subsequently with 10 s far red light (735 nm, 200 µmol photons m-2 s-1). PF, DF and MR transients were simultaneously recorded in the first 1 s illumination with red light, and MR signals were still detected in the following 10 s far red illumination. The redox state of PSI reaction center under continuous light can be detected by monitoring 820 nm reflection [40]. ΔMR/MR0 was calculated according to the relative difference of 820 nm reflection between the maximal oxidized and reduced PSI reaction center [40, 56]. PF transients were quantified by JIP test to calculate Fv/Fm, Vk, RC/ABS, ETo/TRo, REo/ETo, PIabs and PItotal [57].
All redox reactions of the photosynthetic electron transport are reversible, and the back electron transfer and charge recombination in PSII reaction center lead to delayed fluorescence emission from repopulated excited chlorophyll [47]. DF signals are recorded in dark intervals for excluding PF interference under the light [47, 57]. In this study, DF signals in microsecond domain were collected at 20 μs after turning off actinic light for constructing DF transients.
Measurements of MDA and H2O2 contents and histochemical detection of H2O2
MDA content was measured by thiobarbituric acid reaction method for indicating lipid peroxidation degree [58]. Leaf tissues (0.5 g) were ground under liquid nitrogen and homogenized in 5 mL 0.1% TCA. The homogenate was centrifuged at 10000 × g and 4 °C for 10 min to collect the supernatant for measuring MDA and H2O2 contents [25]. Leaves were vacuum-infiltrated with 0.1 mg ml-1 3, 3-diaminobenzidine in 50mM tris-acetate solution (pH, 3.8) and incubated at room temperature in the dark for 24 h. Thereafter, the leaves were decolorized by immersion in boiling ethanol (80%) for 10 min and photographed [59].
Isolation of thylakoid membranes and western blot
As with the method of Yan et al. [28], thylakoid membrane proteins were extracted from the leaves, separated by a 12% (w/w) SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk for 1h, the membranes were incubated with primary anti-PsbA and anti-PsaA antibodies respectively and then incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (PhytoAB, USA). The BeyoECL Plus substrate (Beyotime Biotechnology, China) was applied to test immunoreaction, and the chemiluminescence was recorded by using a Tanon-5500 cooled CCD camera (Tanon, China).
Observation of chloroplast ultrastructure
Similar to Oustric et al. [60] with small modification, leaf pieces (1 mm2) were sampled, fixed in 2.5 % glutaraldehyde in 100 mM phosphate buffer (pH, 7.2) for 2 h at room temperature and washed with the same buffer. The samples were post-fixed in 1 % osmic acid in 100 mM phosphate buffer (pH, 7.2) at room temperature for 4 h, parched through a graded ethanol series (50–100%) and embedded in Spurr’s epoxy resin. Ultra-sections (70 nm) were prepared by using an ultramicrotome (Leica ultracut R, Germany) and stained with uranyl acetate and lead phosphate. A transmission electron microscope (JEM-1230, Japan) was used for observing chloroplast ultrastructure.
Statistical analysis
One-way ANOVA was carried out by using SPSS 16.0 (SPSS Inc., Chicago, IL, USA) for all sets of data. The values presented are the means of measurements with five replicate plants, and comparisons of means were determined through LSD test. Difference was considered significant at P < 0.05.