Plant materials and treatment design
Nandou 12, which is a major soybean cultivar that is closely planted or intercropped with other crops in agriculture production in Southwestern China, was selected as the experimental material. Soybean seeds were provided by the Nanchong Institute of Agricultural Sciences, Sichuan Province, China. All soybean seeds were covered with wet filter paper for 24 hours at 30 ℃. After that, the germinated soybean seeds were grown in containers with a seedling spacing of 10 cm. The size of each container was 15 cm in height, 40 cm in length, and 20 cm in width, and filled with humidified organic soil.
The experiment was divided into two parts. One part was used for morphology measurement. The containers including germinated seeds were directly placed under different light environment treatments, and each treatment included three containers.
The other part was used for physiological and proteomic analyses. The difference in soybean growth period under the various light environments was avoided by growing soybean under normal ambient condition, until the complete development of the first trifoliate leaf. Then, plants were subjected to four different light treatments just after the appearance of the second trifoliate. Furthermore, after 15 days of treatments, all samples were collected at 10 am for the analysis of physiological parameters and differentially expressed proteins. Plants were grown in growth chamber with natural solar radiation, with the temperature maintained at 25 ℃in the day and 20 ℃ during the night. Additionally, the humidity was maintained at 60%, and plants were irrigated with nutrients solution (0.2 % Hoagland's solution) after every couple of days.
The black nylon gauze and Far Red (Fr)-LED was used according to our previous knowledge , to control the PAR and Red-Far Red ration in soybean canopy. The intensity of Fr light in soybean canopy is 5.98 ± 0.14 µmol·m-2·s-1 under dark condition. The PAR and spectral irradiance of soybean canopy were measured at noon on a sunny day, every measurement was replicated five times. The following four treatments were used (Table 2): normal light, normal light plus Fr light, low light, and low light plus far-red light. Every treatment was repeated three times in three different growth chambers. The PAR and spectral irradiance were measured using LI-190SA quantum sensors (LI-COR Inc., Lincoln, NE, USA) and a fiber-optic spectrometer (AvaSpec-2048; Avantes, Netherlands) placed at 10 cm above the soybean canopy, respectively  .
Here Insert Table 2
The plant height from the soil surface to the growing point of soybean. The biomass and leaf area of five soybean seedlings were measured every 14 days after 14 days of sowing under four treatments. Leaves were scanned using a flatbed scanner (CanoScan LiDE 200, Canon Inc., Japan), and the leaf area (cm2) was measured by Image J 1.45 s. Biomass samples were over-dried at 105 ℃ for 0.5 h to stop metabolic processes of tissues and then dried at 80 ℃ for 72 h to a constant weight .
As previously published by Yang et al. , the leaf segments (2 mm × 2 mm) of second trifoliate were placed at 4 ℃ in 3 % glutaraldehyde, and treated with 1 % osmium tetroxide. Then, the fixed segments of soybean leaves were dehydrated and embedded in a graded acetone series and Epon812, respectively. The semithin sections were stained and cut with a diamond knife. Then, the samples were stained with acetate and lead citrate, and observed using a transmission electron microscope (TEM; HITACHI, H-600IV, Japan).
Measurements of sucrose and starch
Leaf samples were over- dried at 105 ℃ for 0.5 h to stop metabolic processes of the tissues and then dried at 80 ℃ for 24 h to a constant weight. According to the methods of Lee et al (2020) with some changes , soluble sugars were extracted from 0.5 g dried samples by homogenization in 5 ml of 80 % (v/v) ethanol. After heating the homogenate in a water bath, the insoluble fraction was removed by centrifugation at 3500 g for 10 min. The precipitate was homogenized and centrifuged again. Supernatants were pooled and then diluted up to 25 ml with 80 % ethanol. The hydrolyzed samples were subjected to centrifugation, and only a small amount (100 uL) of supernatant was collected and added with 100 uL of 30 % Potassium Hydroxide solution followed by boiling, about ten minutes. The solution was cooled down, and the anthrone agent was added. After heating the solution again at 40° for 15 min, it allowed to cool, and absorbance was checked at 620 nm. The amount was calculated using the standard solutions of sucrose. The leftover material is centrifuged tube was utilized for starch extraction, by adding 2 mL of water. Then, the tubes were placed in boiling water bath for 15 min. After cooling, 2 ml of 9.2 M perchloric acid (PCA) was added. After stirring for 15 min, the supernatants were collected after centrifuging the contents at 3500 g for 10 min. The residues were re-extracted two times with 2 ml of 4.6M PCA. After centrifugation, the supernatants were combined, volumes were made to 50 ml with water. Starch was determined colorimetrically using the phenol-sulphuric acid method, as described by .
Photosynthesis, photosynthetic pigment concentration, and quantum yield of PS II
As described by Yang et al. , the second trifoliolate leaf was selected to measure photosynthetic characteristics using a Li-6400 portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA), environment temperature 25 ℃ and a CO2 concentration of 400 µmol mol-1 from 9:00 to 11:00. Eleven light intensity levels (0, 20, 50, 100, 150, 200, 400, 600, 800, 1000, and 1200 µmol m-2 s-1) were imposed. On a light response curve, PPFD was located on the horizontal axis and Pn was on the vertical axis (Pn-PPFD curve). The Pmax and LSP were then estimated using the method proposed by Yang et al. . Chlorophyll fluorescence parameters were obtained using a CI Imager chlorophyll fluorescence imaging system (Technologica Ltd, Colchester, UK). Before each measurement, we placed soybean leaves of each treatment under dark conditions for 10 min. Then actinic illumination (750 µmol m-2 s-1) was switched on, and saturating pulses were applied at 20 s intervals for 15 min. From each of these, the maximum fluorescence (F′m) and the steady-state fluorescence (Fs) were determined in the light condition. The quantum efficiency of the photosystem II was calculated according to the formula (F′m- Fs)/ F′m .
After that, four-leaf disks of 15 mm diameter were obtained from the center of each leaf and then cut into pieces of 3 mm. These leaf-disks were placed in 10 ml of 80% acetone in the dark at 20 ℃ for 24 h. The chlorophyll contents were determined by following a previously published procedure . Triplicates were prepared for each treatment.
Protein extraction, digestion, and iTRAQ labeling
After two weeks of treatment, the second leaf of soybean plants was ground using liquid nitrogen. Following the method of Yang et al. , the powder then shifted to a centrifuge tube. It fragmented using lysis buffer and 1% protease suppressor cocktail under the cold condition with the help of a high-intensity ultrasonic processor (Scientz). The leftover was preceded by centrifugation. Proteins were sedimented under cold using 15% TCA for 2 h at -20 ℃. Following centrifugation at 4 ℃, the afloat was disposed of, and the leftover sediment was rinsed three times using cold acetone. The protein concentration was then determined by dissolving the protein in the buffer. The reduction and alkylation of protein solution were done for 45 min using 20 mM IAA at room temperature in the dark. 100 mM TEAB was then used for the dilution of the protein sample. At last, for the digestion of protein, trypsin was added using the trypsin-to-protein ratio. Approximately 100 μg of protein was digested with trypsin in each sample for further experiments. For iTRAQ labeling, peptides were desalinized with the help of Strata X C18 SPE column (Phenomenex), vacuum-dried, replenished in 0.5 M TEAB, and then operated using 4-plex iTRAQ kit manual instructions.
HPLC fractionation and LC-tandem mass spectrometry (MS/MS) analysis
As previously reported , high PH reverse-phase HPLC divided the sample into fractions by using Agilent 300 Extend C18 column. Following the dehydration through vacuum centrifugation, the peptides were dissolved in 0.1% Formic Acid and loaded onto a reverse-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). EASY-nLC 1000 UPLC system was used to perform Gradient elution at a constant column flow rate of 350 nl/min. The resulting peptides were then analyzed using an Orbitrap FusionTM TribridTM mass spectrometer (Thermo Fisher Scientific).
Database search and analysis
MaxQuant, with an integrated Andromeda search engine (v.188.8.131.52), was used to process the MS/MS data. iTRAQ 8-plex was used for the quantification, and the default values of all the other parameters in MaxQuant were selected, as reported by Yang et al. . To identify the down-regulated or up-regulated protein expression, we used 0.77- or 1.3-fold cut-off with a P <0.05, respectively. GO annotation was used to annotate the proteins . The differentially accumulated proteins were also assigned to the KEGG database .
Proteomics reliability was confirmed by the help of qRT-PCR assay. RNA isolation was done according to the protocol of Yuan et al. . In our study, the β-tubulin gene was taken as a reference control. RT-PCR was done on a CFX96 system machine (Bio-Rad, USA). The primers used are listed in the table S2.
Significance was calculated using one-way ANOVA by computer-based program SPSS (version 16.0). Data are given as mean ± standard deviation from three replicates. Statistical significance was recognized at P < 0.05.