The experiment was conducted from 2020 to 2021 at Chongzhou experimental site of Sichuan Agricultural University, Sichuan, China (30°33^′N, 103°39^′E). The daily average maximum and minimum temperature from sowing to harvest were 27.0 and 19.0°C in 2020, respectively (Figure 1A). The daily average maximum and minimum temperature from sowing to harvest were 26.8 and 17.8°C in 2021, respectively (Figure 1B). The annual sunshine and precipitation were 1,161.5 h and 1,012.4 mm, respectively. The field soil was a light loam with pH 7.1, 24.3 g/kg organic matter, 1.6 g/kg total N, 1.3 g/kg total P, 15.2 g/kg total K, 299.5 mg/kg available-P, and 169.4 mg/kg available-K.

The field experiment was conducted using the two-factor randomized block design, with planting patterns (maize–soybean strip intercropping and monocropping) as the main factor whereas soybean cultivars as the secondary factor. The variety of maize was zhenghong-505 (ZH-505, semicompact plant) and soybean cultivars included Chuandou-16 (CD-16, shade-intolerant plant) and Tianlong No. 1 (TL-1, shade-tolerant plant). The classical wide and narrow row planting was adopted in maize–soybean strip intercropping system (Yang et al., 2014; Cheng et al., 2020), with 2 m of each strip width and 6 m of each strip length (Figures 2A,C). Regardless of strip intercropping or monoculture, the planting density of TL-1 and CD-16 was 20 plants/m², and the planting density of ZH-505 was 10 plants/m². In the monocropping system, the same planting way as strip intercropping soybean without maize strip was conducted as the control (Figures 2B,D). The maize and soybean with few differences in growth period were directly broadcast in the field in the form of seeds. The area of each individual treatment plot was 12 m² for both strip intercropping and monocropping, with a row × plant spacing of 40 cm × 20 cm for maize and a row × plant spacing of 40 cm × 10 cm for soybean. Each treatment that includes six biological repetitions was randomly planted in the whole experimental field. As base fertilizer for maize, 80 g/m² of compound fertilizer (N: P: K = 15: 15: 15) was applied before sowing. The urea (N ≥ 46%) of 7.8 g/m² and 13.2 g/m² was applied at jointing stage and heading stage, respectively (Cheng et al., 2020), whereas soybean did not apply fertilizer in the whole growth period because of fertile experimental site.

After 35 days of sowing, the samples of nine soybean plants with uniform and continuous growth for each treatment were collected every 14 days until harvest, with a total of five times. These samples were used for physiological and biochemical analyses. The collecting data information included the lodging rate (LR), LRI, PH, SD, LC, and activity of 4CL and PAL, canopy structure parameters that include LAI, MLA, and TC. Additionally, the Pn of the inverted trifoliate (PnIT, net photosynthetic rate), the average Pn of the whole plant (APnWP), and also stem anatomical structure that includes vascular, phloem, xylem, and pith were measured at R1 stage (early blooming stage). Grain yield and yield composition that include seed yield, full-pods, non-full-pods, and branches were measured at mature stage.

Lodging rate (Eq. 1) was calculated by randomly investigating a soybean strip without damage for each treatment according to the previous method (Cheng et al., 2020). LRI (Eq. 2) from the third to fifth internodes at the base of soybean stem, as an important and comprehensive stem strength index, was measured as reported earlier (Hussain et al., 2020a,b).

where the LR represents the lodging rate of soybean in a plot, the TNLPP represents the total number of lodging soybeans in a plot, and the TNPP represents the total number of soybeans in a plot.

where the LRI represents the lodging resistance index of soybean stem, SBR represents soybean stem bending resistance, the MSL represents the main stem length of soybean, and the AGW represents the above-ground biomass fresh weight of soybean.

The LAI, MLA, and TC of soybean canopy of each treatment were measured by Digital Plant Canopy Imager (CI-110, Zealquest Scientific Technology Co., Ltd., China), which measured four uniform measuring points in every treatment between two rows of soybean and took three more diagonal cells for repetition.

Between 9:00 a.m. to 11:00 a.m. on a sunny day, the PnIT and APnWP were measured with the portable photosynthetic analyzer LI-COR 6400 (Li-Cor Inc., Lincoln, NE, United States) at R1 stage of soybean. Totally, five plants with uniform growth were selected from each treatment and three repetitions, according to the earlier reported research (Cheng et al., 2020).

The stem anatomical structure that includes vascular, phloem, xylem, and pith were measured according to the previous method (Rajput et al., 2018) with slight modification. The third internode of soybean stem was kept in FAA fixing solution for one month to soften. According to the following Table 1, the stem samples were dehydrated and transparent. Next, the stem samples were processed according to the following steps. ➀ Soaking wax: the paraffin wax (melting point: 57–63°C) was put into a beaker and melted it in a water bath pan (DXY-5H, Shenzhen Dingxinyi Experimental equipment Co., Ltd., China), in which the samples were soaked for about 8 h. ➁ Slicing: the wax block with stem samples was fixed on the slicer (RM 2245, Leica Microsystems Ltd., Wetzlar, Germany), and the slice thickness was set to 10 μm and sliced automatically (the blade was changed frequently). ➂ Exhibition: the cut samples were unfolded in a water bath pan (40°C), then placed on the slide glass (marked), and put the slide glass on the baking machine (HI 1210, Leica Microsystems Ltd., Germany) at 40°C for drying. ➃ Dewaxing: in a ventilated place, the slide glasses containing the samples were dissolved in a container containing xylene for 30 min and dried. ➄ Dyeing: the safranin was dropped on the stem samples for 10 min, slowly removed by distilling water, and dried naturally. Then, the fixed o-fast green dye was dropped on the treated samples for 10 s, slowly removed by distilling water, and dried naturally. ➅ Decolorization and observation: the slide glasses containing the samples were put in 75% alcohol to remove the residual dye and observed with a microscope (M205 FA, Leica Microsystems Ltd., Wetzlar Germany) after dried naturally. The final result is shown in Figure 3.

A total of 15 soybean plants with uniform and continuous growth were selected for each treatment. The number of full-pods, non-full-pods, and branches per plant, and also grain yield per unit area of soybean, were measured after natural drying. Likewise, 15 maize plants with uniform and continuous growth after natural drying were selected to measure grain yield per unit area.

The stem length of soybean plant was regarded as PH, which was measured with flexible rule. The SD in the middle of the third internode of the stem was measured with the Vernier caliper. The LC, 4CL, and PAL from the third to fifth internode of the stem were determined by the test kits (Lignin, 4CL and PAL Test Kits, Suzhou Grace Biotechnology Co., Ltd., China) according to the previous methods (Cheng et al., 2020).

Shading-tolerance coefficient (Eq. 3) was used to describe the changes in target traits value of soybean between intercropping and monocropping, respectively. The CSTC (Eq. 4) was used to objectively evaluate the shade tolerance of soybean in intercropping systems (Li et al., 2014).

where the STC represents shading-tolerance coefficient. The j represents different target traits of soybean. The TTV_i and TTV_m represent target traits value of soybean in monocropping and intercropping, respectively. When the value of STC is large, which means the target trait of soybean is less affected by shading; otherwise, it is more affected by shading.

where the CSTC represents CSTC. The n represents the number of target character of soybean. The j represents different target traits of soybean. When the value of CSTC is large, which means in the low-light environment, soybean shows a strong shade tolerance; otherwise, it possesses a weak shade tolerance (Liang and Li, 2004; Li et al., 2014).

All data were collected and stored by Microsoft Excel 2019 software. The Adobe Photoshop 2020 software was used to draw the maize and soybean plants and image typesetting. The Image Pro Plus software was used to process and calculate the areas of stem cross-section, vascular, phloem, xylem, and pith, etc. The LRI, LC, Pn, and the areas of stem cross-section, vascular, phloem, xylem, and pith and also seed yield, full-pods, non-full-pods, and branches were subjected to two-way ANOVA using the Origin Pro 2021 software. Significant differences among means of every treatment were separated according to Fisher’s LSD at the level of p ≤ 0.05. The t-test at p ≤ 0.05 was applied for correlation analysis. The LR, PH, SD LAI, MLA, and TC were subjected to one-way ANOVA to assess the effects of shading of maize and variety difference in this study.

In the strip intercropping condition, the seed yield, the number of full-pods, and branches of CD-16 and TL-1 were significantly lower than those of monoculture, whereas the number of non-full-pods was the opposite (Table 2). The seed yield of TL-1 was 13.5% higher than that of CD-16 in the monocropping, whereas 21.8% higher than that of CD-16 in the strip intercropping (average of 2 years) (Table 2). A number of full-pods, non-full-pods, and branches of TL-1 were 16.6, 17.0, and 9.0% higher than those of CD-16, respectively, in the monocropping (Table 2). Similarly, in the strip intercropping, a number of full-pods, non-full-pods, and branches of TL-1 were 26.6, 22.4, and 29.6% higher than those of CD-16, respectively (Table 2). However, there was no significant difference between monoculture and intercropping in maize yield. The result showed that, compared with CD-16, the low decrease in grain yield of TL-1 in strip intercropping was due to its relatively high the number of full-pods and branches.

Different soybean cultivars and planting patterns had significant impacts on LR (Figure 4A) and LRI (Figure 4B) of soybean. We noticed that whether CD-16 or TL-1, the lodging only occurred in the strip intercropping system, and with the development of soybean and maize growth, the LR increased continuously (Figure 4B). However, the LR of TL-1 was significantly lower than that of CD-16 (38.2% lower on average), and the lodging time of TL-1 in the field was approximately 14 days later than that of CD-16 (Figure 4B). To explain these phenomena, we further measured the LRI of soybean and found that the LRI of soybean in the strip intercropping was significantly lower than that of monocropping regardless of CD-16 or TL-1 (Figure 4A). Furthermore, the LRI of TL-1 was 30.0% lower than that of CD-16 in monocropping; however, but it was 15.1% higher than that of CD-16 in the strip intercropping (Figure 4A). This phenomenon could be explained according to the Eq. 2. Consequently, in the maize–soybean strip intercropping system, TL-1, a shade-tolerant plant, had relatively strong stem strength compared with CD-16, which was beneficial to maintain the upright growth of the plant.

There was no significant difference in the LAI between TL-1 and CD-16 before soybean shaded by maize (0–49 days after sowing) in the strip intercropping (Figures 5A,B). Thereafter, the LAI of TL-1 was significantly lower than that of CD-16 during the whole later growth period of soybean, irrespective of the cropping system (Figures 5A,B). Notably, the LAI of TL-1 in the monocropping was 20.01% lower than that of CD-16, whereas that of TL-1 in the strip intercropping was only 16.0% lower than that of CD-16 (average of the last four sampling times). Additionally, the peak time of LAI of TL-1 and CD-16 in strip intercropping (63 days after sowing) was approximately 14 days earlier than that in monoculture (77 days after sowing), due to the shading stress of maize on soybean (Figures 5A,B).

Irrespective of the cropping system, the TC of TL-1 was significantly higher than that of CD-16 during the whole growth period of soybean (Figures 5C,D). After shaded soybean by maize, the TC of TL-1 and CD-16 in the strip intercropping was lower than that of in monocropping (Figures 5C,D). It was noteworthy that the TC of TL-1 in the monocropping was 10.91% higher than that of CD-16, but the TC of TL-1 was 11.42% higher than that of CD-16 (average of the last four sampling times) in the late maize shading.

Whether it was in strip intercropping or monoculture, the MLA of TL-1 was significantly higher than that of CD-16 from 0 to 49 days after sowing, whereas that of TL-1 in the strip intercropping was lower than that of CD-16 from 49 to 63 days after sowing (Figures 5E,F). Immediately, the MLA of T-1L and CD-16 decreased rapidly in the strip intercropping after the 63rd day of sowing, but the decreasing rate of TL-1 was faster as compared to CD-16 (Figures 5E,F). What should be of concern was that the MLA of TL-1 in the monocropping was 20.55% lower than that of CD-16, whereas the MLA of TL-1 was only 4.15% lower than that of CD-16 (average of the last four sampling times) in the late maize shading.

In a word, the shading of maize decreased the LAI and TC, but increased the MLA of intercropping soybean canopy, regardless of CD-16 or TL-1; however, the variation range of these canopy parameters of TL-1 were lower as compared to CD-16. The result showed that in the strip intercropping system, the canopy spatial structure of shade-tolerant plants (TL-1) was slightly changed by the shading stress, which was conducive to increasing light energy capture and the photosynthesis of leaves at different canopy levels in a low-light environment.

Regardless of CD-16 or TL-1, the PnIT and APnWP of strip intercropping soybean were significantly lower than those of monoculture soybean at R1 stage (Figures 6A,B). Furthermore, the PnIT of TL-1 was obviously lower than that of CD-16 (14.0% lower on average), irrespective of planting patterns (Figure 6A). However, compared with CD-16, most of the leaves of TL-1 in both strip intercropping and monocropping maintained a relatively high level of Pn, which led to a higher APnWP of TL-1 (12.3% higher on average) (Figure 6B). The result showed that in the strip intercropping system, the shade-tolerant plant (TL-1) which was not easy to change in spatial structure could maintain the relatively high Pn of leaves at different canopy levels.

Compared with monoculture, maize shading reduced the SD but increased the PH of strip intercropped soybean regardless of CD-16 or TL-1, but the change range of SD and PH of TL-1 was smaller than that of CD-16 (Figures 7A–D). However, the SD of TL-1 in the strip intercropping was even higher than that of CD-16 as compared to monocropping (Figure 7A).

Anatomical structure of stems showed that planting patterns, soybean cultivars, and their interactions have significant effects on the number of vascular bundles and the area ratio of vascular/stem, phloem/stem, xylem/stem, and pith/stem at R1 stage (Table 3 and Figure 3). The number of vascular bundles and the area ratio of phloem/stem and xylem/stem of TL-1 in the monocropping were significantly lower than those of CD-16 (Table 3 and Figure 3). In addition, for TL-1, the percentage of decline in the number of vascular bundles between monoculture and intercropping was 4.08%, whereas for CD-16, the percentage of decline was 19.50% (Table 3). In contrast, those anatomical structure parameters of TL-1 in the strip intercropping were significantly higher than CD-16 (Table 3 and Figure 3).

Consequently, in the maize–soybean strip intercropping system, there were relatively high number of vascular bundles and the area of vascular, phloem, xylem, and pith in shade-tolerant plant (TL-1) compared with CD-16, which was beneficial to maintain a relatively thick stem.

As compared to monoculture, regardless of CD-16 or TL-1, maize shading decreased the activity of PAL (Figure 8A) and 4CL (Figure 8B) of strip intercropped soybean, which led to the LC of intercropping soybean decreasing (Figure 8C). Furthermore, compared with CD-16, the TL-1 had lower PAL and 4CL activities and lower LC in monoculture (lower than 22.1, 21.4, and 21.0%, respectively), but it was opposite in the strip intercropping (higher than 116.4, 30.6, and 12.0%, respectively) (Figures 8A–D). Consequently, in the maize–soybean strip intercropping system, there was a relatively high activity of PAL and 4CL in shade-tolerant plant (TL-1) compared with CD-16, which was beneficial to maintain a relatively high LC of stem.

To demonstrate the effect of plant canopy structure on stem morphology and yield components of soybean, we further analyzed the correlation between the canopy structure of plants and the key morphological and physiological parameters of stem and also yield, etc. (Figure 9). The result showed that the LAI was significantly and positively correlated with seed yield, full-pods, and branches, but significantly and negatively correlated with PH and non-full-pods (Figure 9). Additionally, the TC significantly and positively correlated with LRI, PnIT, APnWP, LC, PAL, 4CL, seed yield, full-pods, and branches, whereas significant negative correlation existed between the TC and MLA (Figure 9). Additionally, there was significant and negative correlation between the MLA and SD, LC, PAL, seed yield, full-pods, and branches (Figure 9). Consequently, these parameters, such as LAI, TC, and MLA, could be potentially used to evaluate canopy structure of plants and reflect the growth status of plants and final yield formation.

The STC of shading-tolerance target traits in TL-1 (e.g., LAI, MLA, SD, PH, APnWP, seed yield, and branches) significantly larger than those of CD-16 (Figures 10A–E,G–I). The STC of PnIT was not significant in TL-1 and CD-16 (Figure 10F). The possible reason is that the decrease of PnIT caused by shading stress is roughly the same, regardless of CD-16 or TL-1. Moreover, compared with CD-16 (CSTC = 0.608), TL-1 (CSTC = 0.716) had a higher CSTC, which indicated that it had a stronger shade tolerance in strip intercropping system.

In the maize–soybean strip intercropping system, soybean was vulnerable to shading stress from maize during the intergrowth, which thus affected its canopy structure and photosynthesis (Hideki et al., 2005; Feng et al., 2019). A large number of studies have shown that crop canopy structure was closely related to interception and utilization of photosynthetic active radiation (Yang et al., 2014; Feng et al., 2016; Charbonnier et al., 2017). In this study, the LAI, MLA, and TC of strip intercropped soybean were significantly lower than those of monoculture (Figure 5). Consequently, a serious impact was that the PnIT and APnWP of strip intercropped soybean were significantly lower than those of monoculture soybean at R1 stage (Figures 6A,B). In addition, the photosynthetic rate of intercropped soybean had been proved to be weaker than that of monoculture soybean (Su et al., 2014; Yao et al., 2017; Fan et al., 2018; Raza et al., 2019), which provided additional evidence to support that the intercropped soybean with lower the PnIT and APnWP experienced a shaded environment. Another important evidence showed that the low-light intensity aggravated senescence of leaves, which results in a decrease in leaf numbers (Lim et al., 2007; Zhou et al., 2019a,b), which in turn weakened canopy structure and photosynthesis of strip intercropped soybean (Figures 5, 6).

Variations in leaf area distributions of different soybean cultivars in the field grown were largely determined by differences in the light intensities at the lower canopy levels, but independent of the angle of leaf inclination (Blad and Baker, 1972). In this study, the shade-tolerant soybean TL-1 had high STC of canopy structure (STC_LAI = 0.799, STC_TC = 0.839, and STC_MLA = 0.949) (Figures 10A–C), which was conducive to maintaining the relative stability of spatial structure of the canopy in low-light environment. Importantly, compared with the shade-intolerant soybean CD-16, the shade-tolerant soybean TL-1 had a more perfect canopy structure such as higher TC and lower LAI and MLA (Figure 5), which would allow greater penetration of light to the lower canopy levels (Blad and Baker, 1972; Sakowska et al., 2018). Therefore, compared with shade-intolerant soybean CD-16, the shade-tolerant soybean TL-1 had a higher APnWP (Figure 6B), even though its PnIT was lower (Figure 6A). As a result, a good canopy structure of soybean is the prerequisite for maintaining high APnWP in low-radiation areas. However, in the intercropping system, the molecular mechanism of low-light environment regulating soybean canopy structure (LAI, TC, and MLA) was still unclear, which needs further research.

Most crops in the intercropping system showed yield advantages as compared to the corresponding monoculture (Li et al., 2001, 2020a; Ghosh, 2004; Chen et al., 2019; Feng et al., 2019). In this study, the soybeans in the strip intercropping system showed no yield advantages as compared to monoculture, irrespective of cultivars, across the two years in field experiments (Table 2). One possible explanation was that the canopy morphological formation of strip intercropped soybean was severely inhibited during the intergrowth with maize, which results in plant lodging (Figure 4B) and yield reduction (Table 2). Correlation analysis showed that the canopy structure of strip intercropped soybean was significantly correlated with LR and yield (Figure 9). A compact-type canopy structure was beneficial to intercept more light energy to promote stem morphogenesis and yield formation (Wang et al., 2017; Li et al., 2020b). In addition, the low dry matter allocation index limiting the yield of strip intercropped soybean might be another reason, because in the reproductive stage, the dense canopy structure with relatively low PAR reduced the number of full-pods and branches (Table 2), which increases the allocation of dry matter to leaves and stems and thus decreases the allocation of photosynthetic products to seeds (Board, 2000; Liu et al., 2010; Cheng et al., 2020). In contrast, the shade-tolerant soybean TL-1 with high STC (STC_{Seed yield} = 0.526 and STC_Branches = 0.397) (Figures 10H,I) had relatively high seed yield, full-pods, and branches in the strip intercropping system (Table 2). This was one of the important strategies to increase the yield of shade-tolerant soybean in areas with low-light radiation.

Crops with the perfect canopy structure were propitious to intercept more light energy to promote stem morphogenesis and yield formation (Board, 2000; Wang et al., 2017; Cheng et al., 2020; Li et al., 2020b), especially in the condition of intercropping. Some studies have shown that crops in intercropping showed strong shade avoidance responses, which include thinner stem thickness, longer stem, and petiole and also lower PAR inside the canopy and photosynthetic capacity (Yang et al., 2014; Liu et al., 2016; Fan et al., 2018). In this study, regardless of shade-intolerant soybean CD-16 or shade-tolerant soybean TL-1, The stem of strip intercropped soybean was obviously weaker than that of monoculture in terms of external morphology (lower SD and higher PH) (Figure 7) or internal structure (less vascular bundles and smaller area ratio of vascular/stem, phloem/stem, xylem/stem, and pith/stem) (Table 3 and Figure 6), which provided additional evidence to support that there was less PAR inside the canopy of intercropped soybean. In addition, regardless of soybean cultivars, some studies (Cheng et al., 2020; Hussain et al., 2021) were consistent with the observations that the strip intercropped soybean with an unfilled canopy showed a lower activity of PAL and 4CL (Figures 8A,B), and also LC (Figures 8C,D) compared with monocropping. Regardless of planting system or cultivars, the stem with low LC was one of the key factors for plant lodging and yield reduction (Lourenco et al., 2016; Hussain et al., 2020a; Figures 4, 8C,D). Additionally, the cellulose content in soybean stem was also closely related to plant lodging in intercropping (Liu et al., 2016). In this study, the canopy structure, LC, stem morphology, leaf photosynthesis, and yield components were also closely correlated (Figure 9). Thus, it could be seen that improving the canopy structure and the LC in the stem during intercropping was beneficial to decrease the lodging risk of soybean.

Fortunately, soybean cultivars of different genotypes had different responses to various shade environments. The shade-tolerant soybean (CSTC = 0.713) slightly changed its spatial structure of canopy and stem morphology and physiology in maize–soybean strip intercropping system (Figures 3, 5, 7). Some studies showed the morphology of shade-tolerant plants had lower plasticity (Gong et al., 2015; Chmura et al., 2017), so as to maintain the canopy structure stability of the plant in low-light conditions. Therefore, compared to the shade-intolerant CD-16, the shade-tolerant TL-1 with a relatively perfect canopy had the stronger external morphology (Figure 7) and internal structure (Table 3 and Figure 3) in the strip intercropping, in favor of increasing LRI and reducing the LR of soybean in the field (Figure 4). Interestingly, for TL-1, the percentage of decline in the number of vascular bundles between monoculture and intercropping was 4.08%, whereas for CD-16, the percentage of decline was 19.50% (Table 3), which shows that the TL-1 could slightly reduce the number of vascular bundles in the stem and maintain the stem strength in strip intercropping (Hussain et al., 2021). Besides, high-light intensity was one of the important abiotic factors to improve photosynthesis of plants (Yang et al., 2014; Xue et al., 2016; Feng et al., 2019; Zhou et al., 2019a,b). As compared to shade-intolerant CD-16, most of the leaves of shade-tolerant TL-1 in both strip intercropping and monocropping maintained a relatively high level of APnWP (Figure 6B), which led to a higher accumulation of photosynthetic outcome in stem (Figures 3, 7, 8 and Table 3) and grains (Table 2). Finally, intercropping could improve the microclimate conditions of coffee plants, such as lowering temperature and irradiance level and increasing air relative humidity (Partelli et al., 2014). Thus, what is the relationship between soybean field microclimate and plant lodging resistance and yield formation in the maize–soybean strip intercropping systems? Which needs to be further explored.