Field experiments was carried out from May 2015 to October 2015 at the experimental farm of Shenyang Agricultural University, Shenyang, Liaoning Province, China, The experiment was laid out a completely randomized block design with two cropping (maize-soybean intercropping and soybean monocropping). The row direction was north–south layout. Soybean and maize were sown on May 3rd, 2015. Soybean cultivar Liaodou32 was used, and maize cultivar used was Zhengdan958. The intercropping used wide-narrow row planting, and the ratio of maize to soybean rows in the intercropping was 2:2. The distance between the maize and soybean was 80 cm, and the distance between two rows of maize or two rows of soybean was 40 cm. The densities of sole cropping soybean, intercropped soybean and intercropped maize were 150000, 150000, and 60000 plants ha^-1. The uppermost and fully expanded leaves were used for measurements at R2 stage (full flowering).

The average PAR and maximum PAR of soybean canopy changes of different cropping were measured in a sunny day using a light meter (AccuPAR LP-80, United States) according to the method of Yang et al. (2014), and listed on Figure 1.

Light response curves of Photosynthesis were measured using a LI-6400XT (Li-Cor, United States). The parameters were measured on uppermost and fully expanded leaves from 09:00 to 11:30 am on a clear day. The temperature and CO₂ concentration of leaf chamber were maintained at 25°C and 380 μmol mol^-1, respectively. PAR was increased from 0 to 1500 μmol photons m^-2 s^-1 (0, 20, 50, 80, 100, 200, 400, 600, 800, 1000, 1200, 1500 μmol m^-2 s^-1, 36 min). And then, after linear fitting, light compensation point (LCP), LSP and light-saturated net photosynthetic rate (A_max), apparent quantum efficiency (AQE) and dark respiration rate (R_d) were estimated by the method of Ye (2007).

Diurnal variation of leaf gas exchange was measured on a clear sunny day. Photosynthesis was measured with a LI-6400XT (Li-Cor, United States) equipped with 2 cm × 3 cm clear chamber. P_n and C_i were recorded at intervals of 2 h from 08:30 am to 16:30 pm. The measured leaves were kept at their natural angle of posture exposing to direct irradiance outside leaf chamber. The temperature and CO₂ concentration of leaf chamber were kept at natural environment.

Light response curves for fluorescence were monitored by PAM-2500 chlorophyll fluorometer (Heinz Walz GmbH, Germany), and according to the method of Chen et al. (2014). Rapid light curves were performed with gradually increasing irradiance in 11 steps with 180 s intervals. For each step, the irradiance is 0, 198, 363, 619, 785, 981, 1160, 1386, and 1663 μmol m^-2 s^-1, and the fluorescence signal was recorded, respectively. The data were recorded and read data from the PamWin V3.12g (system control and data acquisition system).

Diurnal variation of leaf chlorophyll fluorescence was measured on a clear sunny day by PAM-2500 chlorophyll fluorometer (Heinz Walz GmbH, Germany). The fluorescence signals were recorded at intervals of 2 h from 08:30 am to 16:30 pm. The measured leaves were kept at their natural angle of posture exposing to direct irradiance outside leaf chamber. Then, the Y(II) and other parameters were calculated as described by Baker (2008).

After a dark adaptation for 30 min, chlorophyll a fluorescence transient (OJIP) of soybean leaves were measured by the plant efficiency analyzer (Hansatech Instruments Ltd., Norfolk, United Kingdom) in a solar day at 10:00 am to 12:30 pm. The uppermost and fully expanded leaves were used for measurements. We obtained the parameters of chlorophyll a fluorescence which could reflect the PSII activity of soybean leaves. Then, the PSII parameters derived from the OJIP transient were analyzed based on the method of Strasser et al. (2004, 2010).

After the measurements described above completed, the leaves were collected for determination of chlorophyll content (Chl a, Chl b, Chl a+b, Chl a/b). Chlorophyll pigments were extracted by grinding leaves in 80% acetone in the dark at room temperature and were expressed as mg/g FW from the equations of Porra (2002). The leaf area was measured by a portable leaf area meter (LI-3100C, LI- COR, United States).

The middle segments of the uppermost and fully expanded leaves were sampled and fixed in a formaldehyde solution (FAA). Leaf segments were dehydrated, cleared and embedded in paraffin. Then these samples were cut by RM2235 rotary microtome (Leica Microsystems Ltd., Germany) at thickness of 10 μm. Sections were stained with Safranin O and Fast green, then observed and captured by Axio Imager A2 microscope (Zeiss, Germany). Leaf thickness, palisade tissue thickness and spongy tissue thickness were quantified by using ZEN imaging software (Zeiss, Germany).

The middle segments of the uppermost and fully expanded leaves were collected at 12:30, and immediately stored in liquid nitrogen, and then kept at -80°C. Leaf sample was homogenized with 50 mM phosphate buffer (pH 7.8) containing 10 mM Polyvinylpyrrolidone (PVP) and 0.2 mM EDTA in an ice bath, and centrifuged at 12,000 × g and 4°C for 20 min. The supernatant was used for MDA and enzyme analysis. The MDA content was assayed by the thiobarbituric acid test (Hodges et al., 1999). Activity of antioxidant enzymes was measured according to Samantary (2002). The activity of superoxide dismutase (SOD) was assayed by measuring its ability to inhibit the photochemical reduction of NBT at 560 nm, and was expressed as units per g of fresh weight. The activity of catalase (CAT) was determined by measuring the decrease of oxidized phenols of H₂O₂ at 240 nm, and the activity of CAT was expressed as units per g of fresh weight during 1 min.

The experiments were arranged in a completely randomized block design with three replications. One-way analysis of variance (ANOVA) and the Duncan’s multiple range tests were used to assess each of the parameters using SPSS statistics software (Version 20, SPSS, Chicago, IL, United States). The graphs were made using Sigmaplot (Version 12, Systat Software).

The light environment of different planting patterns was showed in Figure 1. The average PAR on the soybean canopy in intercropping was significantly lower than those in monocropping. The maximum PAR on the soybean leaf was significantly lower than those in monocropping in early morning and late afternoon, but was exposed to high radiation at noon.

Leaf in intercropping showed a significantly higher photosynthetic pigment concentration per fresh weight, and significantly lower chla/b than those under monocropping (Table 1). The leaf area per plant in intercropping was significantly lower than that in monocropping.

In contrast to soybean grown in monocropping, the leaf became thinner, and the thickness of both leaf and palisade tissue were significantly decreased, however, the spongy tissue thickness was little changed.

P_n increased rapidly as PAR increased to 600 μmol⋅m^-2⋅s^-1 and then increased slowly to saturation (Figure 2). P_n under intercropping was higher than that in monocropping at low PAR, while lower at high PAR. A_max (light-saturated net photosynthetic rate) of soybean leaf in intercropping was about 18.96 μmol⋅m^-2⋅s^-1, it was only about 65.79% of A_max in monocropping (28.82 μmol⋅m^-2⋅s^-1, Table 2). The LCP, LSP, and R_d (dark respiration rate) in intercropping were lower than those in monocropping, while AQE was higher than those in monocropping.

Results obtained from rapid light curves showed that Y(II) (quantum yield of photochemical energy conversion in PS II), qP (coefficients estimating the fraction of open PS II reaction centers based on a puddle model), and qL (coefficients estimating the fraction of open PS II reaction centers based on a lake model) were decreased gradually with the increase of PAR (Figures 3A,C,E). And Y(II), qP and qL in intercropping were higher than those in monocropping. ETR (electron transport rate) increased significantly with the increase of PAR, and ETR in intercropping saturated at lower PAR than those in monocropping (Figure 3B). NPQ (non-photochemical quenching) and Y(NPQ), expressed the thermal dissipation of excitation energy, had a significant rise with the increase of PAR (Figures 3D,F).

P_n increased with the increase of light intensity, and reached maximum at 10:30, and then began to decrease. P_n in intercropping was significantly lower than that in monocropping (Figure 4A). C_i and Y(II) decreased with the increase of light intensity, and reached minimum at noon, and then began to recover. C_i in intercropping was significantly higher than that in monocropping. Y(II) in intercropping was significantly lower than that in monocropping at 10:30–14:30 (Figures 4B,C). NPQ increased with the increasing of light intensity, and reached maximum at noon, then began to decrease. And NPQ in intercropping was significantly higher than that in monocropping at 10:30–14:30 (Figure 4D).

At noon, the NPQ (non-photochemical quenching) and qN (coefficients of non-photochemical quenching) in intercropping were significantly higher than those in monocropping, while qP (coefficients estimating the fraction of open PS II centers based on a puddle model) and qL (coefficients estimating the fraction of open PS II centers based on a lake model) in intercropping was significantly lower than those in monocropping (Table 3).

At noon, Y(II) in intercropping was significantly lower than those in monocropping, while Y(NPQ) in intercropping was significantly higher than those in monocropping. Y(NO) in intercropping was lower than those in monocropping, but there was no significant difference between them (Table 4).

The fluorescence parameters derived from fast fluorescence kinetic are listed in Table 5. At 10:00 am, φ_Po (maximal quantum yield of primary photochemistry), ψ_Eo (efficiency/probability that an electron moves further than Q_A^-), φ_Eo (quantum yield for electron transport), PI_ABS (performance index on the absorption basis) and W_k (the ratio of variable fluorescence at the K-step to the fluorescence difference F_j-F_o) in intercropping were significantly higher than those in monocropping, while δ_Ro (efficiency/probability with which an electron from the intersystem electron carriers is transferred to reduce end electron acceptors at the PSI acceptor side) in intercropping was significantly lower than those in monocropping. At midday (12:30 pm), ψ_Eo, φ_Eo, δ_Ro, and φ_Ro in intercropping became lower than those in monocropping, while W_k in intercropping were higher than those in monocropping.

The MDA content and activity of antioxidant enzymes were showed in Table 6. The MDA content, activities of SOD and CAT in intercropping were significantly higher than those in monocropping at noon.

In intercropping system, high crop significantly reduced the PAR for soybean, and soybean had to make some response to adapt the change of light environment. The decrease of LCP, LSP, and A_max (light-saturated net photosynthetic rate) in intercropping indicated that the photosynthetic capacity was limited. The increase of AQE indicated that the ability of light-intercepting gets promoted in light-limited environment conditions, and this was beneficial for higher light utilization efficiency in intercropping. The increase of R_d (dark respiration rate) indicated that soybean in intercropping dropped the energy expenditure. All these features contributed to the efficient interception and absorption of light and carbon gain in intercropping. And these were similar to that plant grew in shade condition (Zhang et al., 2004; Tateno and Taneda, 2007; Gong et al., 2014). Therefore, the shade of maize leaded to the decrease of photosynthetic capacity of soybean leaf in intercropping. And the shade-tolerant and high photosynthetic efficiency soybean cultivar could be choosed to improve the photosynthetic capacity and yield of soybean in intercropping (Liu et al., 2014; Cui et al., 2015).

The decrease of photosynthetic capacity was caused by stomatal or non-stomatal limitations (Gong et al., 2015). Previous study suggested that the decrease of photosynthetic capacity of spring barley in shade condition was not caused by stomatal effect (Zivcak et al., 2014). Our result showed that P_n was limited in intercropping, however, C_i inside the leaf in intercropping was higher than that in monocropping (Figure 4). This research showed the same result that the decrease of P_n in intercropping was not caused by stomatal effect.

Leaf photosynthetic rate is related to chlorophyll content (Shao et al., 2014). Chl a is essential for determining photosynthesis, and Chl b determine the wavelengths of light that can be absorbed by the organism (Field et al., 2013). Intercropped soybean leaf contained more chl a and chl b content per weight and had lower chl a/b than those in monocropping, which could broaden the wavelengths of light that could be absorbed, and effectively increase the ability of light capture (Gong et al., 2014). This is an important adaptation for plants growing in shaded environments.

The leaf and palisade tissue thickness of soybean leaf in intercropping became thinner, which resulted in the reduction of chloroplast, where carboxylation reactions of photosynthesis take place, mostly located in palisade tissue (Terashima et al., 2006; Gong et al., 2015). Therefore, thinner palisade tissue in intercropping decreased the photosynthetic capacity of soybean leaf.

The higher PI_ABS (performance index on the absorption basis) and φ_Po (maximal quantum yield of primary photochemistry) in intercropping indicated that the light-intercepting capacity and PSII activity was enhanced. But the δ_Ro (the efficiency/probability with which an electron from the intersystem electron carrier s was transferred to reduce end electron acceptors at the PSI acceptor side) and φ_Ro (the quantum yield for the reduction of the end electron acceptors at the PSI acceptor side) of the plants grown in intercropping were lower than those of the monocropped plants (Table 5). And this indicated that the quantum efficiency from PSII to PSI in intercropping were lower than that in monocropping, electron transport between Q_B and PSI and the acceptor side of PSI might be inhibited (Wang et al., 2006; Li et al., 2014; Li L. et al., 2016; Zivcak et al., 2014). Intercropped soybean plants increased the photochemical efficiency of PSII, but the electron transport was limited and the accepted capacity of PSI was low. This was one of the reasons that soybean growth was inhibited and showed a low photosynthetic capacity in intercropping. Therefore, the limitation of electron transport and the changing of morphology of soybean leaf in intercropping were the reason that the photosynthetic capacity of soybean cultivars decreased.

As shown in Figure 1B, intercropped soybean leaf were exposed to high radiation at noon, and had to take a series of reactions to adapt it. Leaf in intercropping exhibited higher Y(II) and lower NPQ than those in monocropping in the morning and afternoon, indicated the higher efficiency of light utilization at low radiation. However, leaf in intercropping showed lower Y(II) and higher NPQ than those in monocropping at noon (from 10:30 to 14:30), this indicated that the absorbed energy of PSII flux to photochemical processes reduced and this part of energy converted into the non-photochemical energy loss or non-photochemical quenching in high radiation. Higher NPQ indicates a higher transthylakoid proton gradient (ΔpH), which leads to more efficient downregulation of electron transport from PSII to PSI, hence, lower risk of hydroxyl radical production on PSI (Joliot and Johnson, 2011; Brestic et al., 2015). These all were beneficial for dissipating excess excitation energy in time and avoiding photo-damage. The lower qL in intercropping suggested that soybean plants grown in intercropping could close or inactivate more reaction centers to limit the energy input into PSII in high radiation (Table 3).

The fate of absorbed light energy was shown in Table 4. Y(NPQ) is an important indicator to reflect photoprotection. In high radiation, Y(II) in intercropping reduced, while Y(NPQ) increased significantly. The significant increase of Y(NPQ) suggested more absorbed energy flux from the photochemical energy conversion to the regulated non-photochemical energy loss in PSII in intercropping in order to adapt high radiation condition. Higher Y(NPQ) implied that there was still photochemical energy conversion or protective regulatory mechanisms to dissipate the light energy absorbed by soybean plants. Y(NO) is an important indicator of photo-damage. There is no significant difference in Y(NO) between intercropping and monocropping, which indicated that wide light-fluctuation in intercropping did not cause photo-damage. The high excitation pressure is considered to be directly related to the photo-damage (Kornyeyev et al., 2010; Zivcak et al., 2014), and is easy to happen at high light. Together with low LSP, A_max, and ETR in intercropping, we could expect severe photo-damage in intercropping. However, there were low differences in photo-damage. One possible explanation is that the photo-protection ability is increased to avoid photo-inhibition with the increasing of excitation pressure at high light (Niinemets and Kull, 2001).

The higher W_k in intercropping demonstrated that the donor side of PSII was seriously inhibited compared to monocropping in high radiation at noon (Chen et al., 2004; Li L. et al., 2016). The higher ψ_Eo and φ_Eo at 10:30 suggested that the quantum efficiencies in PSII electron transfer chain of soybean plants grown in intercropping were enhanced compared to the monocropped soybean. At 12:30, with the effect of high radiation, the ψ_Eo and φ_Eo in intercropping and monocropping decreased; the ψ_Eo and φ_Eo in intercropping were lower than those in monocropping. The higher decrease of parameters ψ_Eo and φ_Eo in intercropping reflects higher light susceptibility to high radiation. These indicated photo-inhibition of soybean leaf grown in intercropping caused a huge accumulation of Q_A^- (Strasser et al., 2004). Excess electrons transported from PSII to the acceptor side of PSI may result in the occurring of photo-inhibition (Huang et al., 2015). Thus, we expected that soybean leaf grown in intercropping was more susceptible to photo-inhibition in high radiation. However, the lower PSII connectivity of shade leaves might keep the excitation pressure lower, physiologically more acceptable level and thus protected photosynthetic apparatus against high light (Zivcak et al., 2014).

MDA content is used as an indicator of lipid peroxidation (Sudhakar et al., 2001; Spicher et al., 2016). In our study, The MDA content of Intercropped soybean leaf was significantly higher than monocropped one. And this indicated that the higher accumulation of ROS led to much more membrane peroxidation within the thylakoid and chloroplasts in intercropping than this in monocropping. The higher excess excitation energy and the lower electron transportation activity between PSII and PSI in intercropping probably turns the photosynthetic apparatus into a stronger ROS source (Gill and Tuteja, 2010; Vanlerberghe et al., 2016). Antioxidative defense mechanisms can scavenge the ROS to protect the photosynthetic apparatus. In our study, intercropping increased the activities of SOD and CAT in soybean leaf to scavenge the higher production of ROS. And this was beneficial for the photosynthetic apparatus to against oxidative stress. Together with no significant difference in Y(NO) between intercropping and monocropping, these suggested that although there was a higher ROS in intercropping, the higher activity of antioxidant enzymes could scavenge the ROS in time to be not causing photo-damage.