Rapeseed Supports Hairy Vetch in Intercropping, Enhancing Root and Stem Morphology, Nitrogen Metabolism, Photosynthesis, and Forage Yield

Abstract

The global shortage of high-quality forage has significantly constrained the development of animal husbandry. Leveraging the complementary effects of forage rapeseed and hairy vetch intercropping can enhance forage yield and quality; however, the underlying mechanisms of overyielding in forage rapeseed–hairy vetch intercropping systems remain unclear. Over two years of field experiments, three cropping systems—rapeseed sole cropping, hairy vetch sole cropping, and rapeseed–hairy vetch intercropping—were investigated to assess the effects of intercropping on root and stem morphology, canopy light distribution, leaf photosynthetic physiology, and nitrogen metabolism. Our results demonstrated that intercropping increased forage biomass and crude protein yield by 14.3–20.0% and 30.7–92.8%, respectively, compared to sole cropping. Intercropping significantly enhanced root biomass, increasing lateral root biomass by 81% compared to rapeseed sole cropping. It also improved stem anatomical traits, including the cortex area (58.8–80.7%), cortex thickness (25.1–38.3%), number of vascular bundles (18.0–37.3%), vascular bundle length (17.8–18.4%), vascular bundle perimeter (6.7–18.7%), vascular bundle area (34.6–63.9%), and stem breaking strength (25.7–76.6%). Additionally, intercropping optimized vertical canopy light interception, reduced the activity of antioxidant enzymes (CAT, POD, SOD) and reactive oxygen species (ROS) accumulation, and enhanced the activities of glutamine synthetase and nitrate reductase, stomatal traits, and photosynthetic rates in the leaves of both crops. Structural equation modeling revealed that, in the intercropping system, improved population lodging resistance directly promoted nitrogen metabolism and leaf photosynthetic rates, ultimately increasing population biomass. In summary, rapeseed–hairy vetch intercropping improved canopy light distribution, strengthened rapeseed stem anatomy and root penetration, and enhanced population lodging resistance, leaf photosynthetic physiology, and nitrogen metabolism, thereby boosting forage biomass and quality. The supportive role of rapeseed in the intercropping system elucidates the overyielding mechanisms of rapeseed–hairy vetch intercropping, offering a theoretical framework for optimizing forage production systems worldwide.

1. Introduction

Lodging is a common production issue that adversely affects crop yield and quality. In grain crops such as rice, wheat, maize, and rapeseed, lodging typically occurs before crop maturity, leading to reductions in yield and quality of up to 40% and 10%, respectively [1,2]. Similarly, in forage crops such as silage maize, ryegrass, vetch, forage rapeseed, and alfalfa, lodging not only limits yield and quality but also increases the incidence of pests and diseases [3]. Moreover, lodging complicates mechanized harvesting, exacerbates harvest losses, and poses risks to animal health [1].

Intercropping, through interspecies interactions such as competition and compensation, has been shown to improve nutrient absorption and resource use efficiency, leading to higher crop yields [4]. Forage rapeseed and hairy vetch are valuable forage resources due to their high biomass and crude protein content. Preliminary findings from our research suggest that rapeseed–hairy vetch intercropping is a promising system for biomass production. The potential mechanisms underlying its benefits include nitrogen fixation by hairy vetch, which makes nitrogen available to non-leguminous crops, improved utilization of environmental resources due to crop complementarity, reduced spread of diseases, and effective suppression of weeds [5]. Within this system, hairy vetch, with its semi-prostrate, soft, and flexible stems, is highly susceptible to lodging. In contrast, rapeseed has stronger stems with greater epidermal hardness and diameter, conferring superior lodging resistance [6]. Given the distinct agronomic and morphological characteristics of rapeseed (Brassica napus L.) and hairy vetch, the overall lodging resistance of the intercropping system may be improved. However, how intercropping influences the lodging resistance of rapeseed, hairy vetch, and the whole system remains poorly understood.

Optimizing light distribution is crucial for improving photosynthesis and energy utilization in crop systems [7]. Improper canopy structure caused by lodging disrupts vertical and spatial light distribution, reduces photosynthetic rates in leaves, and inhibits lignin and cellulose accumulation in stems, ultimately diminishing crop yield and quality. Enhancing lodging resistance within crop populations is therefore essential to constructing optimal canopy structures and achieving high light-use efficiency. In intercropping systems, stronger-stemmed crops can serve as supporting crops, providing structural stability for lodging-prone crops [8]. Additionally, intercropping often combines crops of differing heights and light preferences, improving light distribution and spatial complementarity within the population [9]. Light interception at different spatial levels is commonly used to evaluate light distribution in intercropping crop populations [10,11]. However, the mechanisms through which rapeseed–hairy vetch intercropping affects canopy structure and light distribution remain unclear.

Roots, leaves, and stems play key roles in crop yield formation, functioning as sources and conduits for plant resources. Intercropping can alter spatial heterogeneity among crops, reshaping canopy structure and photosynthetic capacity [7,12,13]. Leaf photosynthesis is closely linked to stomatal conductance and nitrogen metabolism [14]. For instance, in maize-soybean intercropping systems, optimized light distribution has been shown to enhance photosynthetic rates by increasing stomatal density and area in leaves [15]. Stem strength, influenced by stem diameter, anatomical structure, and cell wall composition, is a critical factor for lodging resistance [16]. Furthermore, root–root and root–soil interactions in intercropping systems promote root penetration and expansion, enhancing water and nutrient uptake. These improvements contribute to increased leaf photosynthesis and nitrogen metabolism. However, the interaction mechanisms among roots, stems, leaf photosynthesis, and nitrogen metabolism in rapeseed–hairy vetch intercropping systems remain unexplored.

To address these gaps, this study was conducted with three treatments: rapeseed sole cropping (SR), hairy vetch sole cropping (SH), and rapeseed–hairy vetch intercropping (RH). The objectives of this study were to elucidate the effects of rapeseed–hairy vetch intercropping on (i) rapeseed lodging resistance, stem anatomical structure, and light interception at vertical; (ii) reactive oxygen species (ROS) content and antioxidant enzyme activity in seedling leaves of rapeseed and hairy vetch; and (iii) photosynthesis and stomatal structure in the leaves of both crops. We hypothesize that the structural support provided by rapeseed in the intercropping system can optimize light distribution and interspecies interactions, thereby improving photosynthetic physiology and nitrogen metabolism, ultimately enhancing forage yield and quality.

2. Materials and Methods

2.1. Experimental Conditions

This study was conducted based on the winter forage crops-forage maize double-cropping long-term field experiments in the experimental field of Huazhong Agricultural University, Wuhan, China (30.52° N, 114.31° E). The data presented in this article were collected from 2020 to 2022. Prior to planting in 2017, the initial soil nutrient contents were as follows: total nitrogen, 0.88 g kg⁻¹; total phosphorus, 0.48 g kg⁻¹; total potassium, 16.09 g kg⁻¹; organic matter, 11.62 g kg⁻¹; alkali-hydrolyzable nitrogen, 96.6 mg kg⁻¹; available phosphorus, 14.2 mg kg⁻¹; and available potassium, 141.7 mg kg⁻¹. During the 2020–2021 and 2021–2022 cropping seasons, the monthly average temperatures gradually decreased from 20 °C to 5 °C between October and January (seedling stage) and then increased from 5 °C to 20 °C between February and April (bolting and flowering stages). The 2020–2021 season experienced higher monthly average rainfall, particularly during the seedling and harvest stages of forage crops, whereas rainfall levels during 2021–2022 were extremely low except for January. Total solar radiation was sufficient during the seedling and overwintering stages of rapeseed in both seasons. However, the flowering and late flowering stages in the 2021–2022 season received higher levels of solar radiation (Figure S1).

2.2. Experimental Design and Crop Management

This study utilized two forage crops: rapeseed (Brassica napus L.) and hairy vetch (Vicia villosa Roth.). Three planting configurations were established: rapeseed sole cropping (SR), hairy vetch sole cropping (SH), and rapeseed–hairy vetch intercropping (RH). Planting densities: sole rapeseed was planted at a density of 375,000 plants ha⁻¹ with a row spacing of 30 cm, while sole vetch was sown at a rate of 37.5 kg ha⁻¹ with the same row spacing. For the intercropping treatment, rapeseed density remained at 375,000 plants ha⁻¹, and the vetch sowing rate was increased to 75 kg ha⁻¹. Hairy vetch was planted between rapeseed rows (row intercropping) with a 15 cm row spacing between the crops. Each treatment was replicated three times, and each plot measured 20 m in length and 2 m in width [5]. Basal fertilization included 600 kg ha⁻¹ of compound fertilizer (N–P–K = 15%–15%–15%) and 15 kg ha⁻¹ of boron fertilizer. Two months after emergence, 112.5 kg ha⁻¹ of urea was applied as a topdressing fertilizer. To ensure uniform plant density, rapeseed seedlings were thinned at the 3–5 leaf stage, while hairy vetch seedlings were thinned after germination to achieve the target density. Hairy vetch plants were grown without inoculation.

2.3. Forage Biomass and Crude Protein Yield

Forage fresh weight was measured by randomly harvesting three replicates of 1 m² from each plot at the final flowering stage of rapeseed. To determine moisture content and biomass, 2.5 kg samples from each plot were dried in an oven. Samples were first heated at 105 °C for two hours to deactivate enzymes, then dried at 80 °C to a constant weight [17].

The dried samples were ground, and 0.2 g subsamples were digested using the H₂SO₄-H₂O₂ ashing method to determine nutrient contents [18]. Nitrogen content (NC) and crude protein content (CPC) were determined using the Kjeldahl method with a Kjeltec Auto Analyzer (Hanonk9860; Hanon Instruments, Jinan, China) as described by [19]. Crude protein yield (CPY) was calculated using the following formula:

CPY = biomass × CPC

(1)

2.4. Root Architecture and Biomass

Six representative rapeseed plants, along with their surrounding soil cubes (30 cm × 30 cm × 30 cm), were carefully excavated from each plot during the flowering stage. Roots were cleaned in mesh bags using running water. Root architecture traits were analyzed using an Epson Expression 1640XL root scanner (300 dpi), and data were processed with WinRHIZO Pro Software Version 2019 (Regent Instruments Inc., Quebec, QC, Canada). After scanning, roots were dried in an oven at 105 °C and then at 80 °C to a constant weight to measure lateral and main root biomass [20].

2.5. Stem Diameter and Anatomical Structure

Stem samples were collected after root sampling. Sections (5 cm long) were taken from the base of the rapeseed stem (10 cm above the ground) and the middle portion (10 cm below the first effective branch). Stem diameter was measured using a vernier caliper. The samples were then fixed in FAA (70% ethanol) solution under vacuum to ensure complete fixation. Paraffin sections were prepared, and 6 μm slices were cut using a Leica RM2235 microtome (Leica Microsystems, Wetzlar, Germany). Sections were stained with 1% Safranin-O and 0.5% Fast Green solution, sealed with neutral gum, and scanned using a Pannoramic MIDI slide scanner. Cell morphology, arrangement, and vascular bundle counts were analyzed using CaseViewer software version 2.1 (Synaptive Medical, Toronto, ON, Canada) [21].

2.6. Light Interception, Gas Exchange, and Stomatal Structure

2.6.1. Light Interception Rate

Light interception rates were measured in sole and intercropping populations using an AccuPAR LP-80 canopy analyzer (Decagon Devices, Inc., Pullman, WA, USA) between 11:00 and 14:00 during the overwintering, budding, flowering, and late flowering stages of rapeseed. Measurements were taken at 10 cm intervals, both horizontally and vertically, from 10 cm above the soil surface to the canopy apex [22].

2.6.2. Photosynthetic Gas Exchange

During the flowering stage, under clear-sky conditions between 10:00 and 15:00, the net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO₂ concentration were measured. Six fully expanded third leaves from the apex of rapeseed plants and functional hairy vetch leaves from the middle canopy of each plot were selected for measurements using a Li-Cor 6800 portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA). For vetch, leaf area was calculated using the ellipse area formula due to the smaller size of vetch leaves. The Li-Cor 6800 chamber was set to a 2 cm² area and equipped with red, green, and blue LED light sources. Parameters included a leaf temperature of 35 °C, light intensity of 1200 μmol m⁻² s⁻¹, a red:blue light ratio of 90:10, CO₂ concentration of 400 μmol mol⁻¹, and relative humidity of 60% [23].

2.6.3. Stomatal Structure

At the site where leaf gas exchange measurements were conducted, a total of 18 leaf samples, comprising six 5 mm × 5 mm leaf disks per plot of rapeseed or vetch, were collected. These samples were thoroughly mixed and then immersed in a 2 mL centrifuge tube containing a 2.5% glutaraldehyde fixative solution. All samples were then stored in a 4 °C refrigerator for fixation for a duration of 6 h. Subsequent dehydration of the leaves was achieved using ethanol solutions with varying concentrations (50%, 70%, and 80%). Three leaves from each tube were chosen, secured to the mounting table with double-sided tape, and coated with a gold sputter. Subsequently, the samples were imaged under a vacuum scanning electron microscope (JEOL JSM-6390LV, JEOL, Tokyo, Japan) at a magnification of ×500, with at least six representative fields of view captured for each leaf sample. Image J software (version 1.53e, National Institutes of Health, Bethesda, MD, USA) was employed to quantify the stomatal density (D) and size (S) of the leaves. The stomatal shape was defined as elliptical, and the stomatal area (S) was calculated by measuring the length (SL) and width (SW) of the guard cell:

$$S={\displaystyle \frac{L}{2}}\times {\displaystyle \frac{W}{2}}\times \pi$$

(2)

The maximum theoretical stomatal conductance (gs,max) was estimated according to [24]:

$$g_{\mathrm{s},\mathrm{m}\mathrm{a}\mathrm{x}}={\displaystyle \frac{d\times D\times a_{max}}{1.6v(1+\frac{\pi }{2}\sqrt{a_{max}/\pi })}}$$

(3)

where d is the diffusion coefficient of water vapor in air; v is the molar volume of air; a_max is the maximum area of the open stomatal pore; and l is the stomatal pore depth in fully open stomata. g_s,max for each leaf was calculated as the sum of g_s,max on the abaxial and adaxial sides.

2.7. Enzyme Activity and ROS (Reactive Oxygen Species) Content

Residual leaf samples were preserved in liquid nitrogen and stored at −80 °C. Leaf tissues were ground in liquid nitrogen using a tissue grinder. The supernatant was extracted via centrifugation (4 °C, 12,000 rpm, 15 min). Reactive oxygen species (ROS) content and antioxidant enzyme activities (SOD, POD, CAT) were quantified using ELISA kits (Jiangsu Meibiao Biotechnology Co., Ltd., Nantong, China). Nitrate reductase (NR) and glutamine synthetase (GS) activities were determined using ELISA kits (Wuhan Keep Biotechnology Co., Ltd., Wuhan, China) following the manufacturer’ s protocols [25].

2.8. Data Analysis

Data were analyzed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Variance among treatments was assessed using the least significant difference (LSD) method. Structural equation modeling (SEM) was performed in AMOS 24.0 (SPSS Inc., Chicago, IL, USA) to explore the effects of intercropping on rapeseed stem breaking strength, nitrogen metabolism, net photosynthetic rates of both crops, and biomass. Path coefficients were derived using maximum likelihood estimation. The SEM model fit was evaluated based on several indices, including the relative chi-square/degree of freedom (X²/df), goodness of fit index (GFI), comparative fit index (CFI), and root mean square error of approximation (RMSEA). Data visualization was conducted using Origin 2021 software (OriginLab Corporation, Northampton, MA, USA).

3. Results and Analysis

3.1. Forage Biomass and Crude Protein Yield

Intercropping significantly influenced forage biomass and crude protein yield (Figure 1). Over two years, results indicated that, compared to sole cropping, intercropping reduced the biomass of rapeseed and hairy vetch by 43.4% and 15.5%, respectively. However, the total biomass under intercropping was 27.5% and 61.9% higher than that of rapeseed and hairy vetch sole cropping, respectively. Similarly, intercropping reduced the crude protein yield of rapeseed and hairy vetch by 14.3% and 20.0%, respectively, but the total crude protein yield under intercropping was 92.8% and 30.7% higher than that of rapeseed and hairy vetch sole cropping, respectively (Figure 1).

3.2. Light Interception in the Vertical Direction and Canopy Height of the Population

Intercropping rapeseed with vetch significantly influenced light interception at various growth stages and vertical canopy heights compared to sole cropping. The canopy height of both sole-cropped and intercropped rapeseed populations increased rapidly from the seedling stage to the flowering stage and then stabilized, while the canopy height of vetch showed minimal change after entering the bolting stage. Compared to the light interception rate of sole-cropped rapeseed and hairy vetch at the same vertical canopy height, intercropping increased the light interception rate of rapeseed and hairy vetch by 5.9–39.4% and 71.6%, respectively, at seedling stage, by 10.6–144.4% and 5.6–71.1% at the bolting stage, by 3.6–129.9% and 57.7–670.9% at the flowering stage, and by 14.6–174.2% and 1.4–41 times at the final flowering stage (Figure 2).

3.3. Root Architecture and Biomass

Intercropping significantly improved root architecture traits, root biomass, and the lateral root ratio compared to sole cropping (Figure 3). The results were consistent across two years. Compared to sole cropping, intercropping increased the root length and surface area of rapeseed by 82.8% and 49.3%, respectively (Figure 3c,d). Additionally, lateral root biomass, main root biomass, and total root biomass were elevated by 86.1%, 19.1%, and 25.8%, respectively, under intercropping conditions (Figure 3f). Intercropping also increased the lateral root ratio of rapeseed by 14.5% compared to sole cropping (Figure 3e).

3.4. Rapeseed Stem Diameter and Breaking Strength

Rapeseed–vetch intercropping significantly influenced the stem diameter and breaking strength of both the basal and middle portions of rapeseed stems during the flowering stage (Figure 4). Intercropping increased the stem diameter of the lower and middle portions by 12.6% and 31.4%, respectively, compared to sole cropping (Figure 4a,b). Similarly, intercropping enhanced the stem breaking strength of the lower and middle portions by 25.7% and 76.6%, respectively (Figure 4c,d).

3.5. Stem Anatomical Structure

Intercropping had a significant impact on the anatomical structure of rapeseed stems, including the cortex area, cortex thickness, number of vascular bundles, vascular bundle length, vascular bundle perimeter, and vascular bundle area, in both the basal and middle portions during the flowering stage (Figure S2). Under intercropping conditions, rapeseed stem tissues were denser compared to sole cropping. For the basal stem, intercropping increased the cortex area, cortex thickness, number of vascular bundles, vascular bundle length, vascular bundle perimeter, and vascular bundle area by 58.8%, 38.3%, 18.0%, 18.4%, 18.7%, and 63.9%, respectively. Similarly, in the middle stem, these parameters were increased by 80.7%, 25.1%, 37.3%, 17.8%, 6.7%, and 34.6%, respectively, compared to sole cropping (

Table 1. Effects of rapeseed and hairy vetch intercropping on rapeseed bottom and middle stem anatomical features.

Year	Stem Position	Treat-ments	CA (μm2)	CT (μm)	VN	VL (μm)	VC (μm)	VA (μm2)
2021	Bottom	SR	6.99 b	0.55 b	34.1 b	0.82 b	1.98 b	0.18 b
		IR	11.35 a	0.75 a	41.3 a	0.96 a	2.35 a	0.30 a
	Middle	SR	2.25 b	0.29 b	18.1 b	0.64 b	1.82 b	0.13 b
		IR	4.08 a	0.38 a	25.7 a	0.78 a	1.96 a	0.18 a
2022	Bottom	SR	7.45 b	0.52 b	34.1 b	0.81 b	2.03 b	0.18 b
		IR	11.58 a	0.73 a	39.2 a	0.97 a	2.41 a	0.29 a
	Middle	SR	2.17 b	0.31 b	18.3 b	0.65 b	1.91 b	0.13 b
		IR	3.91 a	0.37 a	24.3 a	0.74 a	2.02 a	0.17 a

Different lower-case letters within a column represent significant differences according to Duncan’s multiple range test (DMRT) at p  <  0.05. CA, cortical area; CT, cortical thickness; VN, vascular bundles number; VL, vascular bundle length; VC, vascular bundle circumference; VA, vascular bundle area; SR, rapeseed in sole cropping; IR, rapeseed in intercropping.

).

3.6. Gas Exchange

Intercropping significantly affected the gas exchange parameters of rapeseed and vetch leaves, including the transpiration rate (E), net photosynthetic rate (A), intercellular CO₂ concentration (C_i), and stomatal conductance (g_s) (

Table 2. Effects of intercropping on gas exchange of rapeseed and hairy vetch leaf at flowering stage.

Year	Crop	Treatments	E (mol m⁻2 s⁻1)	A (µmol m⁻2 s⁻1)	Ci (µmol mol⁻1)	gs (mol m⁻2 s⁻1)
2021	Rapeseed	SR	0.0043 b	26.1 b	252.0 a	0.311 b
		IR	0.0053 a	29.9 a	261.8 a	0.440 a
	Hairy vetch	SH	0.0023 b	12.1 b	234.2 a	0.130 b
		IH	0.0027 a	19.1 a	217.6 a	0.188 a
2022	Rapeseed	SR	0.0042 b	25.1 b	235.7 b	0.298 b
		IR	0.0050 a	29.1 a	260.3 a	0.428 a
	Hairy vetch	SH	0.0021 b	11.6 b	228.2 a	0.128 b
		IH	0.0026 a	18.6 a	211.5 a	0.181 a

Different lower-case letters within a column represent significant differences according to Duncan’s multiple range test (DMRT) at p  <  0.05. SR, rapeseed in sole cropping; IR, rapeseed in intercropping; SH, hairy vetch in sole cropping; IH, hairy vetch in intercropping.

). For rapeseed leaves, intercropping increased E, A, C_i, and g_s by 21.2%, 15.2%, 7.1%, and 42.5%, respectively, compared to sole cropping. In vetch leaves, intercropping enhanced E, A, and g_s by 20.5%, 59.1%, and 7.1%, respectively, while C_i was reduced by 43.1% compared to sole cropping.

3.7. Stomatal Structure

Rapeseed–vetch intercropping significantly influenced the stomatal morphometric and physiological traits of rapeseed and vetch leaves, including stomatal length (SL), stomatal width (SW), stomatal density (D), stomatal size (S), stomatal index (SI), and theoretical maximum stomatal conductance (g_s,max). For rapeseed leaves, intercropping increased SL, SW, D, S, SI, and g_s,max by 8.9%, 4.4%, 24.8%, 11.3%, 44.8%, and 27.4%, respectively, compared to sole cropping (Figure 5). In vetch leaves, intercropping increased SL, SW, S, and SI by 8.2%, 7.4%, 16.2%, and 12.9%, respectively (Figure 6). However, intercropping did not significantly affect the stomatal density or g_s,max of vetch leaves.

3.8. Glutamine Synthetase and Nitrate Reductase Activity

Intercropping significantly enhanced the activity of glutamine synthetase (GS) and nitrate reductase (NR) in the leaves of both crops during the flowering stage (Figure 7). Compared to sole cropping, intercropping increased GS and NR activity in rapeseed leaves by 19.6% and 31.9%, respectively, and in vetch leaves by 72.0% and 42.1%, respectively. These results suggest that intercropping significantly improved nitrogen metabolism in both rapeseed and vetch leaves.

3.9. CAT, POD, SOD Enzyme Activity and ROS Content

Intercropping effectively reduced the activity of antioxidant enzymes—catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD)—and the reactive oxygen species (ROS) content in the leaves of both crops during the flowering stage (Figure 8). In rapeseed leaves, intercropping reduced the activity of CAT, POD, and SOD by 10.1%, 30.1%, and 13.3%, respectively, and decreased ROS content by 7.5% compared to sole cropping. Similarly, in vetch leaves, intercropping reduced CAT, POD, and SOD activity by 27.5%, 44.1%, and 23.4%, respectively, and ROS content by 12.3%.

4. Discussion

4.1. Intercropping Improves Population Lodging Resistance and Light Interception

In the rapeseed–vetch intercropping system, rapeseed stems and branches provide physical support for vetch, effectively mitigating the lodging issues characteristic of vetch monocropping and elevating the canopy height of vetch. This result aligns with previous studies showing that intercropping vetch with wheat and other crops can reduce lodging risks [6]. Similarly, intercropping systems involving pea, oat, and triticale have demonstrated the potential to reduce lodging in forage crops while improving forage biomass and quality [6,26].

Previous research has suggested that intercropping can alter stem composition. For instance, in soybean–corn intercropping, shading caused by corn reduces the transport of starch to soybean stems, which can inhibit cellulose and lignin synthesis, thereby weakening stem strength [27]. Cellulose and lignin are key components of stem strength [28]. In the rapeseed–vetch intercropping system, the improved light irradiance for vetch may stimulate cellulose and lignin synthesis in vetch stems, fundamentally enhancing their mechanical strength and reducing lodging risks [29]. However, the physical support provided by rapeseed stems likely plays the dominant role in stabilizing vetch plants [6].

Our findings show that intercropping significantly enhances the light interception rate across different canopy heights (Figure 2). In vetch monocropping, the population canopy height remains below 40 cm throughout the growth period, resulting in significant shading of lower leaves and minimal light penetration. Despite the higher leaf area index (LAI) in vetch compared to rapeseed, only the upper leaves receive sufficient sunlight. In contrast, under intercropping, vetch vines climb rapeseed plants, improving ventilation and light distribution within the vetch canopy. This increases light exposure and intensity for the lower leaves of vetch compared to monocropping, consistent with previous findings [30].

In rapeseed monocropping, the shedding of lower leaves during later growth stages causes severe light leakage and reduces light use efficiency significantly (Figure 2). However, in the intercropping system, the presence of vetch enhances light interception at various canopy heights, particularly in the 50–110 cm range, improving light use efficiency and laying a foundation for yield enhancement. The LAI of the intercropped population is a critical factor influencing light interception [31]. Intercropping significantly increases the population LAI compared to monocropping, which is the primary reason for the increased light interception rate [32]. This improvement in light interception throughout the crop growth period is likely one of the key factors driving the increase in biomass production.

4.2. Intercropping Improved Leaf Gas Exchange and Stomatal Structure

Our results demonstrate that rapeseed–vetch intercropping significantly enhances the photosynthetic gas exchange capacity of functional leaves in both crops during the flowering stage. This observation aligns with previous studies on pea–oat intercropping systems [33]. Leaf photosynthetic capacity is primarily influenced by stomatal conductance, mesophyll conductance, and CO₂ carboxylation efficiency [34]. Stomatal conductance plays a direct role in regulating gas exchange across cell membranes, and intercropping significantly increases leaf stomatal conductance, contributing to higher net photosynthetic rates. Similar results were reported in corn–peanut intercropping systems, where enhanced photosynthetic capacity was linked to increased yields [35].

Intercropping also significantly increases stomatal density, stomatal size, and the stomatal index in rapeseed and vetch, which are pivotal factors for the observed improvement in stomatal conductance. Additionally, the altered growth habit of vetch in intercropping (from sprawling to upright) reduces mutual shading, improves light exposure, and increases stomatal conductance and photosynthetic rates [36].

Shading effects in intercropping systems can further influence photosynthesis. While excessive shading reduces photosynthetic rates and stomatal opening [37], mild shading (e.g., approximately 30%) has been shown to enhance photosynthesis and biomass production [38]. Moreover, shading of certain leaves can trigger compensatory photosynthesis in unshaded leaves, as observed in corn [39].

Nutrient uptake also plays a critical role in leaf photosynthesis. Our previous study demonstrated that intercropping rapeseed and vetch significantly enhances potassium (K) uptake in both crops [5]. Potassium is essential for photosynthesis and stress tolerance and directly regulates stomatal function [40,41]. Higher potassium content in intercropping systems may optimize stomatal structure and function, thereby enhancing stomatal conductance and photosynthetic rates [42]. Additionally, improved vascular bundle development in rapeseed stems under intercropping facilitates nutrient and water transport, further contributing to increased photosynthesis [43].

When subjected to biotic or abiotic stress, plants accumulate ROS, which cause damage to cells and tissues. At the same time, antioxidant system enzymes (POD, CAT, SOD) are activated to degrade ROS [44]. In our study, compared to monocropping, both ROS levels and antioxidant system enzymes activities in the leaves of rapeseed and hairy vetch were reduced under intercropping. This suggests that the decrease in ROS content in the rapeseed–vetch intercropping system may not be solely due to the activity of antioxidant enzymes but rather due to the down-regulation of ROS production during the formation stage. Our study also revealed that intercropping significantly increases the activity of glutamine synthetase (GS) and nitrate reductase (NR) during the flowering stage, consistent with findings from maize based intercropping systems [45]. These enzymes play key roles in nitrogen metabolism and protein synthesis [46], influencing nitrogen use efficiency. Both ROS content and nitrogen metabolism are indicators of plant senescence [47]. By reducing ROS content and increasing the activity of nitrogen metabolism enzymes, intercropping may delay the senescence process. Structural equation modeling (SEM) indicated that rapeseed stems positively affect nitrogen metabolism in both crops, indirectly enhancing net photosynthetic rates and gas exchange (Figure 9).

4.3. Effect of Intercropping on Lodging and Forage Yield

Lodging resistance is a critical determinant of rapeseed yield and quality, typically assessed using the lodging index and lodging angle [48]. Our results suggest that intercropping significantly improves stem diameter, stem breaking strength, and root biomass while slightly reducing plant height in rapeseed, collectively contributing to improved lodging resistance.

Detailed anatomical analysis revealed that intercropping enhances cortex area, cortex thickness, vascular bundle number, vascular bundle length, and vascular bundle area in rapeseed stems, explaining the observed improvements in stem strength [21]. Stem anatomical traits were significantly enhanced by the application of N and P fertilizers [29,49]. In the rapeseed–hairy vetch intercropping system, the N and P concentration in rapeseed were significantly improved, which may be one of the key factors contributing to the improvement of rapeseed stem anatomical traits under intercropping conditions [5]. Additionally, interspecific root interactions in intercropping systems induce root development, increasing lateral root biomass and spatial distribution [50,51]. Lateral roots play a critical role in nutrient and water absorption and anchoring plants in the soil [52,53]. Strong root systems support leaf photosynthesis and stem development, which are crucial for reducing lodging risks. Intercropping also enhances soil fertility, water use efficiency, and nutrient uptake, forming the basis for higher biomass and yield [54]. Moreover, under conditions of soil nutrition deficiency, intercropping stimulates microbial diversity and root biomass allocation, particularly the distribution of fine roots. This further improves nutrient absorption, enhances biomass and crude protein yield, and contributes to yield stability [55,56].

SEM results further highlight that stem breaking strength, nitrogen metabolism, and net photosynthetic rate directly or indirectly influence biomass formation (Figure 9). These findings suggest that improved stem quality, nutrient metabolism, and photosynthesis are the primary mechanisms by which intercropping enhances forage biomass and crude protein yield, while simultaneously reducing lodging risks.

5. Conclusions

The intercropping of rapeseed with vetch significantly enhances rapeseed stem strength and root anchoring capacity, providing physical support for vetch and thereby reducing lodging risk. This intercropping system improves canopy light distribution, stomatal structure, and leaf photosynthesis, optimizing the growing environment for both crops. Furthermore, intercropping reduces the activity of antioxidant enzymes and reactive oxygen species (ROS) content in leaves, delays plant senescence, and enhances the activities of nitrogen metabolism enzymes, contributing to improved photosynthetic efficiency. In summary, rapeseed serves as a critical “bracket crop” in the rapeseed–vetch intercropping system by improving canopy light interception, nitrogen metabolism, stomatal development, and photosynthesis, while mitigating the harmful effects of ROS on leaves. These combined effects ultimately lead to increased forage biomass and crude protein yield, offering a sustainable and efficient production strategy for forage crops. Our research focused on the physical supporting effects of the rapeseed stem. Further studies should investigate the effects of belowground interspecific root–root interactions on canopy photosynthesis, lodging resistance, and yield formation in intercropped populations.