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. Seeds were soaked in wet filter paper for 1 day at 30 ℃. The germinated seeds were planted in containers (40 cm in length, 20 cm in width, and 15 cm in height) filled with humidified organic soil with a seedling spacing of 10 cm.
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. Avoiding the differences in soybean growth period under different light conditions, when the soybean growth stage was the development of the first trifoliolate leaf (before the second trifoliolate leaf appeared) under normal light condition, the seedlings were then divided into four groups under different light environment treatments. After 15 days of treatment, the second trifoliolate leaf was sampled around 10 am to measure its physiological parameters and analyze differentially expressed proteins. The plants were grown in growth chamber with natural solar radiation. The soybean seedlings were watered every 2 days with 0.2% Hoagland’s solution . The temperature was maintained at 25 ℃ for 12 h at daytime and at 20 ℃ for 12 h at night. Relative humidity was approximately 60%.
According to our previous report , black nylon net was used to adjust the light intensity (PAR) and Fr light-emitting diode (LED) (36 W, light peaking at 735 nm) light sources were used to adjust the R/Fr ratio in 50 cm height of soybean canopy. 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 and Fig. 8): 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  .
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 destroy the tissues and then dried at 80 ℃ for 72 h to a constant weight .
As described by Yang et al. , the segments (2 mm × 2 mm) of the second trifoliolate leaf were fixed at 4 ℃ in 3% glutaraldehyde, and treated with 1% osmium tetroxide. Then, the fixed segments were dehydrated in a graded acetone series and embedded in Epon812. The semithin sections were stained and cut with a diamond knife. Then, the sections were stained with acetate and lead citrate, and examined 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 destroy the tissues and then dried at 80 ℃ for 24 h to a constant weight. According to the methods of Rosa et al (2009) 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 100 μL supernatant was mixed with 100 μL of 30% KOH and boiled for 10 min. After cooling, 3 mL of anthrone reagent was added to the mixed solution, which was then incubated at 40 °C for 15 min. Absorbance was recorded at 620 nm and calculated using sucrose as the standard. For starch extraction, the residues left in the centrifuge tubes after sugar extraction were added with 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 . Sucrose and starch were 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. . In addition, the quantum efficiency of the photosystem II was calculated according to the method by Yang et al. .
Then, four 15 mm diameter disks were removed from the center of each middle leaf and then cut into pieces with the size of approximately 3 mm. These pieces were placed in 10 ml of 80% acetone in the dark at 20 ℃ for 24 h. The photosynthetic pigment concentrations were calculated according to the method of Sims and Gamon . Triplicates were prepared for each treatment.
Protein extraction, digestion, and iTRAQ labeling
After 15 days of treatment, the second trifoliolate leaves were ground in liquid nitrogen. As described by Yang et al. , the cell powder was transferred into centrifuge tube and sonicated in a lysis buffer and 1% protease inhibitor cocktail on ice by using a high-intensity ultrasonic processor (Scientz). The remaining debris were removed by centrifugation. Proteins were precipitated with cold 15% TCA for 2 h at -20 ℃. After centrifugation at 4℃, the supernatant was discarded, and the remaining precipitate was washed three times with cold acetone. The protein was then redissolved in buffer, and protein concentration was determined. The protein solution was reduced and alkylated for 45 min with 20 mM IAA at room temperature in the dark. The protein sample was diluted by adding 100 mM TEAB. Finally, trypsin was added at a trypsin-to-protein for digestion. Approximately 100 μg of protein for each sample was digested with trypsin for the subsequent experiments. For iTRAQ lambelling, Peptides were desalted with Strata X C18 SPE column (Phenomenex), vacuum-dried, reconstituted in 0.5 M TEAB, and then processed according to the manufacturer’s protocol for the 4-plex iTRAQ kit.
HPLC fractionation and LC-tandem mass spectrometry (MS/MS) analysis
As our previous report , the sample was divided into fractions through high pH reverse-phase HPLC by using Agilent 300 Extend C18 column. After the peptides were dried through vacuum centrifugation, and then were dissolved in 0.1% FA and directly loaded onto a reverse-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). Gradient elution was performed at a constant flow rate of 350 nl/min on an EASY-nLC 1000 UPLC system. The resulting peptides were then analyzed using an Orbitrap FusionTM TribridTM mass spectrometer (Thermo Fisher Scientific).
Database search and analysis
MS/MS data were processed using MaxQuant with integrated Andromeda search engine (v.188.8.131.52). As described by Yang et al. , iTRAQ 8-plex was selected as the quantification method, and the default values of all the other parameters in MaxQuant were selected. A 1.3- or 0.77-fold cut-off with a P <0.05 was used to identify the up-regulated or down-regulated protein expression, respectively. Proteins were functionally annotated using Gene Ontology (GO) annotation. The differentially accumulated proteins were also assigned to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database .
Real-time quantitative polymerase chain reaction (qRT-PCR) verification
qRT-PCR assay was used to confirm the reliability of the proteomics. RNA isolation was performed following the methods of Yuan et al. . The β-tubulin gene was used as the reference control in the present study. RT-PCR was performed on a CFX96 system machine (Bio-Rad, USA). All primers are listed in Table S2.
Significance was determined by one-way analysis of variance on SPSS software (version 16.0). Data were presented as mean ± standard deviation from three independent biological replicates. Statistical significance was considered at P < 0.05.