The experiment was carried out at the Modern Agricultural Science and Technology Demonstration Park (37°56′26″N, 109°21′46″E), Yulin, Shaanxi, China in 2020. The area has a semi-arid continental monsoon climate with an average annual rainfall of 400 mm and temperature of 10°C. The soil in the 0–20 cm layer at the experimental site had a loess-like loam texture and contained 7.34 g kg^–1 organic matter, 0.46 g kg^–1 total nitrogen, 0.75 g kg^–1 total phosphorus, 32.7 mg kg^–1 available phosphorus, 17.88 g kg^–1 total potassium, and 72 mg kg^–1 available potassium with a pH of 8.6.

This study used two mung bean (V. radiata L.) cultivars with different growth periods, namely Xilv 1 (loose-type, the growth period is 85–90 days, plant height is 45 cm, pitch number of main stem are 9–10, pods number per plant are 25–35, identified by the National Identification Committee of Minor Grain Crops, provided by Northwest A&F University) and Yulv 1 (erect-type, the growth period is 90–105 days, plant height is 60 cm, pitch number of main stem are 11–14, pods number per plant are 35–45, bred by Hengshan Daming mung bean system, provided by the Yulin Academy of Agricultural Science). The plants were covered with different densities of polyethylene black shade net to establish the non-shading treatment with natural light conditions of 1,500 μmol m^–2 s^–1 (S0), moderate-shading treatment with low-light conditions of 750 μmol m^–2 s^–1 (S1), and severe-shading treatment with stress light conditions of 375 μmol m^–2 s^–1 (S2). A square iron frame with a height of 2.0 m was used. The light intensity on a sunny day was measured with a light meter (AccuPAR LP-80, WA, United States). Experiments were arranged as a randomized block design with three replicates, and mung beans were planted in the north-south row direction. Each plot area was 10 m² (5 m × 2 m), and mung bean seeds were sowed on May 25 with row spacing of 40 cm and plant spacing of 25 cm. When mung beans grew to the branching stage, shading treatments were imposed. Chemical fertilizer inputs (60 kg N ha^–1 and 75 kg P₂O₅ ha^–1) were conducted in accordance with the traditional methods of local farmers. During the growth period, no additional fertilizers or irrigations were applied. The mung bean was sampled at 30 days for shading treatments to determine the effects of shading condition on experimental parameters.

The top third fully expanded trifoliate leaves of mung bean were taken at 9:00–11:00 for the determination of physiological and molecular parameters. Leaf samples were immediately homogenized in liquid nitrogen and transported from the field to the laboratory. Then, they were stored at −80°C until use for the evaluation of Chl concentration, enzyme activities, metabolite concentration, plant hormone, and key gene expression. Moreover, the aboveground parts were cut and oven-dried at 75°C for 48 h to measure the biomass. All samples were mixed with three mung beans from the plot, and the measurement was conducted three times.

Illuminance (lux) was measured with a ZDS-10 illuminometer (Shanghai, China), and air temperature and relative humidity were determined using a Hygro-Thermometer Psychrometer (DHM2, Tianjin, China). All measurements were taken from 11:00 to 13:00 on cloudless days.

Three mung bean plants from each plot (total 9 plants) were selected randomly to measure agronomic traits. The plant height was measured with a ruler. The stem diameter and first internode length were determined using a vernier caliper. The leaf area was measured with a YMJ-C leaf area meter (Zhejiang, China).¹ By using software analysis (Intelligent leaf area measurement system, Zhejiang Top Cloud Agricultural Science Co., LTD, Hangzhou, China), the leaf area was obtained by taking pictures of the fresh leaves on a whiteboard.

The top third fully expanded leaf from each treatment was used to analyze various photosynthetic characteristics with a CIRAS-3 photosynthesis system (PP Systems, Amesbury, MA, United States) at 9:00–11:00. Gas exchange parameters, such as net photosynthetic rate (Pn), transpiration rate (E), stomatal conductance (g_s), and intercellular CO₂ concentration (Ci), were determined under 1,000 μmol m^–2 s^–1 light intensity, 400 μmol mol^–1 atmospheric CO₂ concentration, and 30°C leaf temperature. Three mung bean plants were used for each plot.

The Chl fluorescence parameters were measured with the MINI-PAM-II fluorometer (Imaging PAM, Walz, Germany). Before measurement, all plants were adapted for 30 min in a dark chamber. The maximal fluorescence of the light-adapted state (Fm) and the maximal PSII quantum yield (Fv/Fm) were determined by a 3,000 ms saturated light pulse. Leaves were illuminated with an actinic light (1,800 μmol (photon) m^–2 s^–1). Fv/Fm, photochemical quenching (qP), and non-photochemical quenching (NPQ) were calculated based on the dark- and light-adapted fluorescence measurements in accordance with the method of Gong et al. (2019).

The total Chl concentration was extracted from frozen samples with 10 ml of 80% acetone in the dark for 24 h. Samples were cut from the middle part with a puncher (1.2 diameter), and the supernatant was measured by a spectrophotometer (UV-2550, Shimadzu, Japan) at wavelengths of 663 and 645 nm to evaluate the Chl a, b, and total Chl concentration (mg/L). Three plant samples were analyzed in each treatment.

Fresh samples (0.2 g) were extracted in accordance with the square method of Berveiller and Damesin (2008). The buffer solution was 0.1 mol L^–1 Tris-HCl (pH 7.4), containing 1.0 mmol⋅L^–1 ethylenediaminetetraacetic acid (EDTA), 7 mmol L^–1 mercaptoethyl alcohol, 10% glycerol, and 1% polyvinyl pyrrolidone (PVP). The supernatant was centrifuged at 15,000 × g for 15 min at 4°C to obtain the enzyme extract. The activity of phosphoenolpyruvate carboxylase (PEP Case) (EC 4.1.3) and Rubisco (EC 4.1.1.39) was measured as described by Bi et al. (2015). The absorbance at 340 nm wavelength was traced by using an ultraviolet spectrophotometer, and the enzyme activity was calculated. The concentrations of ATP, starch, sucrose, and soluble sugar in mung bean leaf were determined and calculated in accordance with the method of Li (2000). Each measurement was repeated three times.

Three middle parts of leaves for each treatment without midribs were taken and fixed in formalin-acetic acid-alcohol solution (ethanol:formaldehyde:glacial acetic acid, 90:5:5). The leaf samples were dehydrated in ethanol solutions and then embedded in paraffin. The tissue sections were co-stained by Safranine and Fast Green and observed with a light microscope (ECLIPSE Ts2, Nikon Instruments Inc., Japan).

Leaf tissue (0.1 g fresh mass) was sampled and added to 1 ml extract (acetonitrile:water, 1:1). The supernatants were extracted on ice for 4 h and centrifuged at 4°C for 12,000 × g for 10 min. An aliquot (800 μl) of the supernatant was purified by solid-phase extraction. The solid-phase extraction cartridges were washed using 1 ml of methanol and equilibrated with 1 ml 50% ACN/H₂O (v/v). The samples were loaded, and then the flow-through fraction was discarded. The cartridge was then rinsed using 1 ml of 60% ACN/H₂O (v/v). Then, the samples were evaporated to dryness under a gentle stream of nitrogen and reconstituted in 100 μl of 10% ACN/H₂O (v/v). All the samples were vortexed for 30 s, sonicated in an ice-water bath for 5 min, and then, centrifuged at 4°C for 15 min at 12,000 × g. The ZORBAX Eclipse XDB C18 column (4.6 mm × 280 mm; 5.0 μm) was used to analyze samples. After the crude extract was purified by reverse-phase solid-phase extraction, ether extraction, and derivatization, the endogenous hormone concentration of mung bean leaf was measured by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS, Agilent Technologies, Ltd., Waldbronn, Germany) with Chromosep C18 column (C18 Sep-Pak Cartridge, Waters Corp., Milford, MA, United States) (Ma et al., 2008; Teng et al., 2010).

The relative expression of VrCRY1, VrCRY2, VrPHYB, VrPIF4, VrEIN3, VrGA2ox, VrGA3ox1, VrGA20ox1, VrROT3, and VrBZR1 was assayed with QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific, MA, United States). The primers for quantitative PCR (qPCR) of genes are shown in Supplementary Table 1. RNA was extracted using the TRIzol™ Plus RNA Purification Kit (Invitrogen, MA, United States). Reverse transcription and amplification of cDNA were performed using SuperScript III First-Strand Synthesis SuperMix for quantitative real-time PCR (qRT-PCR) (Invitrogen).

ANOVA was conducted to analyze data using SPSS (version 19.0 Chicago, IL, United States). Comparisons among different treatments were based on Duncan’s test at 5% probability level. The significance of treatment effects, varieties, and their interactions were assessed using ANOVA with the standard split-plot design method. All graphs were plotted using Origin 2018.

As shown in Figure 1, shading stress significantly decreased the illuminance (p < 0.05). Compared with S0, S1, and S2 were reduced by 56.0 and 80.2%, respectively. However, no significant differences were observed in the air temperature between the control and shading stress condition, and the average values of S0, S1, and S2 treatments were 28.5, 28.9, and 28.4°C, respectively. For relative humidity, the shade condition increased by 11.7% in S1 and 13.8% in S2, and the differences were significant between the control and shading stress (p < 0.05). Through analyzing microclimate factors, we found that the illuminance and relative humidity were greatly affected by shading stress in the field condition.

Shading stress had significant effects on the plant morphological characteristics (Figures 2, 3). Plant height and the first internode length were increased, and the stem diameter, pitch number of main stem, branch number of main stem, aboveground biomass, and leaf area were decreased by shading stress, especially in the S2 treatment. For average two shading treatments (S1 and S2), compared with S0, the plant height, stem diameter, pitch number of main stem, first internode length, branch number of main stem, and aboveground biomass of Xilv 1 under low-light condition were influenced by 48.9, 16.5, 19.4, 13.6, 8.0, 26.4, and 23.3%, and those of Yulv 1 were influenced by 20.4, 5.8, 16.8, 6.3, 14.6, 17.5, and 19.2%, respectively. However, the effect of cultivars × shading treatment interaction on the morphological characteristics was not significant (p < 0.05) (except for the plant height and leaf area). The results showed that greater morphological plasticity represent greater adaptability to shade.

The gas exchange parameters of the two mung bean cultivars are shown in Figure 4. The net photosynthetic rate (Pn), transpiration rate (E), and stomatal conductance (g_s) were significantly decreased by 22.9, 16.5, and 27.4% in Xilv 1 and 9.6, 12.1, and 12.6% (p < 0.05) in Yulv 1 under shading stress (averaged S1 and S2) compared with the control. Greater reduction of photosynthetic parameters was observed in S2 treatment than in S1 treatment. By contrast, the shading stress significantly increased intercellular CO₂ concentration (Ci) of leaves (31.4 and 8.4% in Xilv 1 and Yulv 1, respectively; p < 0.05). Yulv 1 had higher photosynthetic capacity, indicating that this cultivar has strong shade tolerance.

Similarly, the Chl fluorescence parameters, such as maximal PSII quantum yield (Fv/Fm), photochemical quenching (qP), and non-photochemical quenching (NPQ) were determined (Figure 5). Fv/Fm was significantly decreased by 4.9% in Xilv 1 and 2.7% (p < 0.05) in Yulv 1 under shading stress compared with control. No significant differences were observed between S1 and S2 treatment. Furthermore, only S2 treatment significantly decreased qP of Yulv 1 (25.4% lower over normal light control) (p < 0.05). Simultaneously, S1 and S2 treatments induced the increase of NPQ in Xilv 1 and Yulv 1 in the field condition.

In the present study, significant differences were observed in PEP Case, Rubisco, ATP, starch, sucrose, and soluble sugar concentrations at different light intensity treatments (Figure 6). Shading stress significantly decreased the activity of PEP Case and Rubisco and concentrations of ATP, starch, sucrose, and soluble sugar. On average, the reduction under weak light stress was 14.5, 7.0, 20.5, 22.9, 24.2, and 22.0% (p < 0.05), respectively, compared with normal light control (S0). Yulv 1 had strong shade tolerance than Xilv 1, as the decrease of PEP Case, Rubisco, ATP, starch, sucrose, and soluble sugar of Yulv 1 (5.9, 6.4, 22.7, 18.4, 22.6, and 17.3%) under low-light and shading stress was less than that of Xilv 1 (23.2, 7.7, 48.5, 27.4, 25.9, and 26.7%). The effect of cultivars on the enzymatic activity and assimilation substances was significant (p < 0.05) (except for Rubisco activity and sucrose concentration).

Different light conditions had a significant effect on the Chl concentration of mung bean plants (Table 1). The concentrations of Chl a, Chl b, and Chl (a + b) of both cultivars under shading stress increased remarkably compared with those in control, while decreased Chl a/b was found (p < 0.05). Light quantity strongly affects the leaf anatomy (Figure 7). We found that in S0 treatment, the mung bean leaf showed clear and compact tissue structure. With decreasing light intensity from S1 to S2, the palisade tissues and spongy tissues were seriously thinner. In Xilv 1, the leaf tissue arrangement was loose and scattered, the shape was irregular, and the gap was large. These findings indicated that shading stress negatively influenced the mung bean leaf tissue size.

As shown in Table 2, the endogenous hormone concentration of mung bean leaf was strongly affected by shading stress. Specifically, the concentrations of auxin (IAA, indole-3-acetic acid), ABA, GAs (such as, GA1, GA3, and GA7), and BR in both cultivars significantly increased by 36.5, 79.6, 55.1, and 31.1% under shading conditions (p < 0.05), while the concentrations of salicylic acid (SA) and ZT significantly decreased by 62.1 and 24.6% (p < 0.05). Interestingly, the opposite trend was observed in the JA concentration of both cultivars under shading conditions: the JA concentration in Xilv 1 was improved, whereas that in Yulv 1 was reduced. In addition, compared with S0, the variation range of IAA, ABA, GA, BR, SA, Zt, and JA concentrations in Xilv 1 under shading stress was 36.6, 131.9, 53.7, 43.2, 62.6, 17.8, and 106.8% (p < 0.05); by contrast, that in Yulv 1 was 35.9, 19.8, 57.1, 13.1, 61.5, 35.8, and 31.3% (p < 0.05), respectively. The effect of cultivars on the endogenous hormone concentration was significant (p < 0.05) (except for GA3 and JA). Through analyzing the variation of endogenous hormone concentration, Xilv 1 was more sensitive and changed more than Yulv 1 under shading stress.

Furthermore, the expressions of two genes involved in cryptochromes (VrCRY1 and VrCRY2), two genes involved in phytochromes (VrPHYB and VrPIF4), one gene involved in ethylene biosynthesis (VrEIN3), three genes involved in GA biosynthesis (VrGA2ox, VrGA3ox1, and VrGA20ox1), and two genes involved in BR biosynthesis (VrROT3 and VrBZR1) were quantitatively analyzed (Figure 8). The relative expressions of VrCRY1, VrCRY2, VrPHYB, VrPIF4, and VrEIN3 in Xilv 1 were remarkably upregulated by shading stress compared with those in control (p < 0.05). However, for Yulv 1, the gene expression levels under two shading stresses showed different trends. In addition, in endogenous hormone biosynthesis, the relative expression levels of all five genes for GA (3) and BR (2) were upregulated under low-light condition (p < 0.05). The expression levels of VrGA2ox, VrGA3ox1, VrGA20ox1, VrROT3, and VrBZR1 in Xilv 1 and Yulv 1 were increased by 1. 49-, 1. 26-, 1. 58-, 1. 17-, and 1.41-fold and 1.83, 1.18, 1.52, 1.28, and 2.17-fold, respectively, in shading stress compared with the S0 treatment. Furthermore, the effect of cultivars × shading treatment interaction on the gene expression was significant (p < 0.05) (except for VrGA20ox1 and VrROT3).

Crop morphology has a certain degree of plasticity, and there is a corresponding adaptation mechanism to different ecological environments (Gong et al., 2015). Many studies have shown that shading promotes the upward growth of stems and petioles but reduces the leaf area of plants (Liu et al., 2017; Jiang et al., 2020). Similar results were observed in our experiment. The plant height and first internode length of mung bean significantly increased under shading conditions, while the stem diameter, pitch number of main stem, and branch number of main stem significantly decreased, resulting in a reduction in aboveground biomass (Figure 2). The lower the light intensity, the greater the influence of the value. These features indicated that the changes in light environment would affect the morphological parameters of mung bean, and in turn, these morphological changes could induce plants to absorb more light energy and reduce the shading stress. Meanwhile, shading not only reduced the light intensity of the environment, but also increased the relative humidity, as shown in Figure 1. This phenomenon may lead to an increase in the occurrence of diseases and insect pests and further weaken the physiological metabolic ability. Crop leaves are sensitive to the light environment, and the photosynthetic capacity is affected by shading stress to a certain extent (Iram et al., 2021). The net photosynthetic rate, transpiration rate, and stomatal conductance were significantly decreased by 16.3, 14.3, and 20.0%, respectively, under shading stress compared with control (Figure 4). By contrast, shading stress significantly increased the intercellular CO₂ concentration of leaves by 19.9% (p < 0.05), indicating that shading stress weakened the net photosynthetic rate of mung bean leaves by non-stomatal limitation. This effect may be associated with the leaf structure changes under shading stress. Lower light environment induced large cell gap, loose cell arrangement, and decreased palisade and spongy tissue thickness in leaves (Figure 7), resulting in decreased chloroplast channel area through which carbon dioxide enters. Consequently, the thickness of leaves and photosynthetic capacity of mung bean leaves are significantly weakened (Feng et al., 2019).

As an internal probe, Chl fluorescence is widely applied in crop science to analyze the relationship between abiotic stress and photosynthetic capacity (Dai et al., 2009). In the current study, the photosynthesis of mung bean was severely damaged by shading stress as shown by the Chl fluorescence parameters (Figure 5). Increased NPQ indicated that the absorbed energy of PSII flux to photochemical processes was reduced under shading stress, and this part of energy was converted into non-photochemical energy loss as heat (Li et al., 2021). However, Fv/Fm was not functionally impaired, even with a significant decrease. The possible reason was that the higher PSII/PSI ratio contributed to compensate for the reduction in the amount of red light, which is required to excite PSII (Iram et al., 2021). Such results may suggest that vulnerability to a lower photosynthetic rate might be linked with changes in multiprotein complexes (PSI and PSII) (Muneer et al., 2014). Moreover, these changes in Chl fluorescence parameters were strongly related to the decreased photosynthetic rate as suggested earlier. Chl can absorb, transmit, and convert light energy and is an important part of the plant photosynthetic system (Gill and Tuteja, 2010). Shading stress increased the Chl concentration in mung bean leaf, such as Chl a, Chl b, and total Chl a + b (Table 1). This phenomenon was beneficial to absorb and capture more light energy from the low-light environment and improve the efficiency of light energy utilization (Wang et al., 2020). However, decreased Chl a/b was mainly observed because shading stress induced the increase in Chl b concentration than Chl a concentration. Studies have demonstrated that Chl a is directly associated with the leaf photosynthesis capacity, while Chl b is located in the photochromatin complex of PSII for trapping diffuse light (Field et al., 2013; Yao et al., 2017b). Increased Chl b concentration can play an important role in enhancing the survival ability of mung bean under shading environment. Furthermore, we investigated the activities of related photosynthetic enzymes and assimilate metabolism. Our results showed that shading stress significantly decreased the concentrations of starch, sucrose, and soluble sugar in the leaves of mung bean compared with control, ultimately leading to the decline in ATP concentration (Figure 6). These results are consistent with the findings of Hussain et al. (2019), suggesting that anatomical and biochemical changes in shaded plants represent ways to cope with low light availability and energy cost of re-fixing CO₂ induced by leakiness under shading (Amiard et al., 2005; Feng et al., 2019). Although the activities of PEP Case and Rubisco were significantly reduced, the diminutive decline may be negligible to a certain extent. Hence, the performances in photosynthetic enzyme activities in shaded plants were potential physiological strategies for decreasing the energy cost under low light availability and keeping the equilibrium of metabolites and energy fluxes between mesophyll and bundle sheath cells (Sales et al., 2018). Taken together, shading remarkably reduced the net photosynthetic rate of mung bean leaves. On the one hand, this phenomenon was attributed to the destruction of the internal structure, such as palisade and spongy tissues; on the other hand, it was significantly related to the Chl and light energy utilization, thereby resulting in the decrease of photosynthetic capacity and changes in morphological parameters of mung bean (Figure 9).

As information transmitters, plant hormones play an important role in regulating the adaptive response of plants. In Arabidopsis seedlings, IAA, GAs, and BR regulate hypocotyl and petiole elongation (Yang and Li, 2017). In the present study, the increments in the concentrations of IAA, ABA, GAs, and BR were observed under shading stress (Table 2). Due to weak light, a large amount of IAA was synthesized in the leaves of mung bean, which was transported to the stem and acted on the epidermal cells, thus promoting the over-elongation of plant height (Procko et al., 2014). The changes of optical signal can directly affect the concentration of IAA through PIFs. PIF4 is one of the most important factors that regulates IAA biosynthesis by binding the promoter of key IAA biosynthesis enzymes YUCs and triggers shade avoidance (Keuskamp et al., 2010; Hornitschek et al., 2012). For the transport of IAA, excessive IAA produced by shading stress upregulated the relative expression of VrPIF4 (Figure 8), so that IAA was transported to the growing center more quickly.

Gibberellins are a kind of hormones that regulate the plant growth and development. Among the physiologically active GAs, GA1, and GA4 are involved in plant photomorphogenesis and closely related to light signals (Alabadí et al., 2004). The bioactive GA in plants is mainly regulated by the syngenetic genes GA20ox and GA3ox and decomposing gene GA2ox (Reinecke et al., 2013). GAs promoted cell elongation by changing the rheological properties of the cell wall. The expression of these genes could regulate the concentration of GAs and then affect stem elongation. Under shading stress, the relative expressions of VrGA2ox, VrGA20ox1, and VrGA3ox1 related to GA synthesis were increased (Figure 8); the concentrations of GA1, GA3, and GA7 in mung bean were significantly improved (Table 2); and the sensitivity of plants to GAs was obviously enhanced. Genetic evidence showed that GAs stimulated cell elongation by destabilizing the GA signaling repressor DELLA proteins, thus releasing the DNA-recognition domain of the transcription factor (Achard et al., 2007).

Brassinolide is a known steroidal plant hormone, which is important for hypocotyl elongation. The relative expression of BR biosynthesis gene VrROT3 was increased under shade conditions, further enhancing BR biosynthesis and promoting VrBZR1 transcription (Figure 8). VrBZR1 and PIFs are interdependent in the regulation of hypocotyl growth, and the degradation of VrPIF4 or the reduction of VrBZR1 activity weakened their functions (Oh et al., 2012). The simultaneous application of IAA and BR had a superimposed effect on promoting axial growth of the lower embryo in hormone interaction (Keuskamp et al., 2011). Brassinosteroid-insensitive 2 in the BR signaling pathway could reduce the DNA binding ability and transcriptional inhibition ability of IAA transcription factor repressor ARF2 through oxidative phosphorylation, thus enabling activated auxin response factors (ARFs) to regulate the expression of downstream genes (Vert et al., 2008). In addition, shading treatment altered the levels of other hormones, for example, increased ABA and decreased SA and ZT concentrations (Table 2). These observations are similar to those of previous studies (Li et al., 2019; Jiang et al., 2021). ABA mainly inhibits the cell division and elongation and affects the growth of plant organs. The increase of ABA concentration in leaves indicated that the senescence process of mung bean was accelerated under shading. ZT is a kind of cytokinin that enlarges cell volume by promoting the cell lateral growth. The changes of ZT concentration under shading were significantly related to the downregulation of ZT synthesis genes or upregulation of ZT degradation genes (Roman et al., 2016). Therefore, these hormonal trends may be a way for leaves to respond to low light stress.

Light is not only the driving force of plant photosynthesis, but also the signal of plant morphological and physiological adaptation to environmental changes (Jiang et al., 2021). In our study, although Xilv 1 showed a higher increase rate in the plant height and first internode length than Yulv 1 under shading stress, Xilv 1 showed lower aboveground biomass than Yulv 1 (Figure 2). This result could be associated with the management of photosynthate allocation under shading stress. Xilv 1 allocated more photosynthetic products to the elongation of the main stem to obtain more light. Meanwhile, the larger leaf area of Yulv 1 is a potential strategy for maintaining photosynthesis (Figure 2). The thylakoid structure and photosynthetic pigment biosynthesis affect the photosynthetic capacity (Yang et al., 2018b). Lower light intensity decreased leaf thickness, palisade tissues thickness, and spongy tissues thickness of leaves. However, compared with Xilv 1, Yulv 1 still maintained a normal cell structure or had less damage to shading stress (Figure 7). This phenomenon might be due to the cell growth and cell layer number in palisade tissues (Kalve et al., 2014). Furthermore, Yulv 1 had higher Chl concentrations (Chl a, Chl b, and Chl a + b) than Xilv 1 under shading stress (Table 1), the effect of cultivars on the Chl concentration was significant (p < 0.05), indicating that Yulv 1 has stronger tolerance to shading, because increased Chl concentrations under shading conditions, especially the Chl b, were beneficial for enhancing the light harvesting in shade-tolerant varieties (Valladares and Niinemets, 2008), as reported by Zhu et al. (2008) that low light-tolerant hybrid rice exhibited a higher content of Chl b when exposed to low light. This conclusion was directly supported by the higher photosynthetic assimilation substances and enzyme activities (Figure 6). This strong physiological metabolism strategy may be one of the potential mechanisms explaining why Yulv 1 maintained higher shading resistance levels than Xilv 1.

The variable light environment regulates the expression of related genes to improve the fitness (Jiang et al., 2020). PIFs are a kind of transcription factor that directly interacts with the photosensitive chromatin downstream. PIF4, PIF5, and PIF7, the members of the PIF family, are involved in the response to shading stress, and PIF4/PIF5 double mutants and PIF7 mutants are significantly inhibited in hypocotyl elongation under low-light environments (Leivar and Quail, 2011; Li et al., 2012). Our data showed that shading increased the relative expression of VrPIF4 in Xilv 1. However, in Yulv 1, moderate shading (S1) treatment significantly upregulated the relative expression of VrPIF4, and the opposite trend was observed in severe stress (S2) treatment (Figure 8). Similar results were found in VrCRY2 and VrPHYB under shading condition. As a photosensitive interaction factor, VrPIF4 can interact not only with photosensitive VrPHYB, but also with cryptochrome VrCRY2. The changes of these genes indicated that the photoreceptors of Xilv 1 were more responsive to shading than those of Yulv 1, and the downregulated expressions of VrCRY2, VrPHYB, and VrPIF4 were beneficial to maintain the plant physiological metabolism of Yulv 1 and ensure plant growth. In addition, the transcription factor EIN3 in the ethylene signaling pathway has been shown to bind to the promoter of PIF3, thereby promoting the expression of PIF3 and regulating hypocotyl length (Zhong et al., 2012). The upregulated relative expression of VrEIN3 in mung bean leaves under shade condition led to the accelerated plant senescence, which further supported the conclusion that Yulv 1 has stronger tolerance and ecological adaptability to shading stress than Xilv 1. However, the current results are limited, mainly because it was only a 1-year field experiment. To make the results more convincing, we will conduct multi-year or greenhouse studies. In addition, a follow-up study (i.e., transcriptomics, metabonomics, and proteomics) should be conducted to obtain further insights regarding the mechanisms underlying the tolerance advantages. Meanwhile, an effective potential strategy for enhancing the plant tolerance and mitigating injure from shading stress should be exported in the future sustainable agricultural production.