Low R/Fr ratio directly affected soybean growth phenotype under normal or low light intensity (Fig. 1A). The plant height of soybean in the N+Fr treatment was significantly higher than that in the N treatment under normal light intensity. By contrast, the plant height in the L+Fr treatment was decreased by 20.8% compared with that of the L treatment in 42 days after sowing (Fig. 1B). After 14 days of sowing, the soybean total biomasses in N+Fr and L+Fr treatments were significantly higher than those in N and L treatments, respectively. The total biomass under normal light (N and N+Fr treatments) were higher than that under low light (L and L+Fr treatments). At 42 days after sowing, the maximum and minimum total biomasses were 2.35 and 0.34 g plant-1 in the N+Fr and L treatments, respectively. Similar trends on biomass were also found at 14 and 28 days after sowing under different treatments (Fig. 1C). In addition, low R/Fr ratio significantly increased the leaf area per plant at 14 and 28 days after sowing under normal or low light intensity, the change in the trends of leaf area per plant were consistent with the total biomass in different treatments (Fig. 1D).
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Changes in chloroplast ultrastructure, sucrose and starch content
When growing in different light environments, the changes in chloroplast ultrastructure were different, the chloroplast size in N and N+Fr treatments were larger than those in L and L+Fr treatment (Fig. 2A). Starch grain (SG) size also exhibited a similar trend. The starch contents in N+Fr and L+Fr treatments were significantly higher than those in N and L treatments, respectively. The maximum and minimum starch content were 92.36 mg/g in N+Fr treatment and 69.19 mg/g in L treatment, respectively. The sucrose content of soybean growing in N+Fr treatment was significantly higher than those in other treatments (Fig. 2B).
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Chlorophyll content, photosynthesis, and quantum yield of PS II
The Chl a, Chl b, and total Chl in normal light condition (N and N+Fr treatments) were significantly lower than those in low light (L and L+Fr treatment) (Fig. 3). The total Chl contents in N+Fr and L+Fr treatments (low R/Fr ratio) were significantly increased by 6.5% and 14.3% compared with those in N and L treatments (normal R/Fr ratio), respectively.
The light response curves of the assimilation rate vs. the photosynthetic photon quanta flux density (PPFD) of four treatments were shown in Fig. 4. The assimilation rates of the four treatments presented significant difference when PPFD was higher than 200 µ mol m-2 s-1 (Fig. 4A). The maximum values of photosynthetic rate (Pmax) and the light saturation point (LSP) appeared in N+Fr treatment compared with those in other treatment. Pmax and LSP decreased by 14.9 and 47.4% under N treatment with respect to the corresponding values under N+Fr treatment. Similarly, Pmax and LSP decreased by 23.8 and 38.2% under L treatment relative to the L+Fr treatment, respectively. The minimum values of Pmax and LSP, which appeared under L treatment, were 5.77 µ mol CO2 m-2 s-1 in the 318.17 µ mol m-2 S-1, respectively (Fig. 4B).
In addition, the quantum yield of PSII is the fraction of light absorbed by leaves for photochemical electron transport. In this study, the quantum yields of PSII under N and N+Fr treatments were significantly higher than that under L and L+Fr treatments. Furthermore, a reduced R/Fr ratio increased the quantum yield of PSII by 15.18% under L+Fr treatment, respectively, with respect to that under L treatment (Fig. 4C).
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Soybean leaf proteomic analysis
The total protein of the soybean leaves was extracted from different treatments, and the protein profiles were explored using the iTRAQ technique. A total of 9890 protein groups were identified, among which 7834 proteins were quantified (Table S1). On the basis of at least > 1.3- of fold change (P < 0.05), among the quantified proteins, we found that 15 proteins were up-regulated, and 41 proteins were down-regulated for N+Fr vs. N; 102 proteins were up-regulated, and 548 proteins were down-regulated in L+Fr vs. N, 180 proteins were up-regulated, and 183 proteins were down-regulated in L vs. N (Fig. 5A).
The differentially accumulated proteins were classified into three groups (cellular component, molecular function, and biological process) on the basis of GO enrichment analysis (Fig. 5B). The main biological functional categories represented were metabolic, cellular, and single-organism processes. According to the molecular functional properties, these proteins were mainly classified into catalytic activity, binding, and structural molecule activity. The subcellular location- annotation information of the identified proteins indicated that chloroplast-associated proteins accounted for 32.1%, 39.1%, and 36.9% of the unique proteins in N+Fr vs. N, L+Fr vs. N, and L vs. N, respectively (Fig. 5C).
To visualize the differences in protein abundance among the N+Fr, L+Fr, L, and N treatments, we used KEGG pathways and visualized as a heat map through a two-tailed Fisher’s exact test. Thirteen different functional categories were selected for analysis. As illustrated in Fig. 6, among the functional categories, C metabolism and photosynthesis-antenna proteins were related to photosynthetic CO2 assimilation. The proteins involved in photosynthesis in N+Fr and L+Fr treatments were down-accumulated compared with those in N treatment, whereas proteins in L treatment were up-accumulated.
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Key protein associated with photosynthesis assimilation of soybean leaves in response to different light conditions
A total of 12 differentially expressed proteins related to photosynthetic CO2 assimilation were detected by iTRAQ analysis under N+Fr, L+Fr, and L treatments compared with those detected in N treatment. Among these differentially expressed proteins, one protein was related to porphyrin and chlorophyll metabolism, two proteins were involved in PS I, four proteins were associated with PS II, three proteins participated in photosynthetic electron transport, and two proteins were involved in starch and sucrose metabolism (Table 1). The expression levels of nine proteins (i.e., Protochlorophyllide reductase [POR], Photosystem I subunit [PsaD], Chlorophyll a/b binding protein 1 [Lhcb 1], Lhcb 2, Lhcb 4, Lhcb 6, PetE, PetF, and Sus) were up-regulated under L treatment compared with N treatment. However, the expression levels of the two proteins (i.e., POR and Lhcb 1) in N+Fr treatment and two proteins (i.e., PsaH and PetH) in L+Fr treatment were down-regulated compared with those in N treatment.
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qRT-PCR results confirming the differentially expressed proteins
To assess the validity of the iTRAQ data, we randomly selected six gene products, including the POR, PsaD, Lhcb 1, PetE, and Sus gene expressed levels, according to differential protein classification for RT-PCR analysis, (Fig. 7). The qRT-PCR results showed that under L treatment, significant increase in the transcript level was observed for POR, PsaD, Lhcb 1, and PetE compared with N treatment. The POR, PsaD, and PetE expression levels were up-regulated under L+Fr treatment. By contrast, the expressions levels of POR, PsaD, and Lhcb 1 were down-regulated under N+Fr treatment compared with those under N treatment. The change in the Gmss 1 was different from that of POR in N+Fr, L+Fr, and L treatments.
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