Potential for Cultivating Various Legume Species in Controlled Environment Agriculture

Abstract

Legumes are among the most important plants capable of biological nitrogen fixation. However, there is a significant knowledge gap regarding the specifics of cultivating legumes in hydroponic systems under controlled environment conditions, particularly nitrogen metabolism at different growth stages, which this study addressed. Chickpeas, faba beans, lentils, soybeans, and sugar peas were cultivated in deep-water hydroponics without rhizobia, with a nutrient solution as the nitrogen source. The legumes displayed significant variations in growth patterns and nitrogen dynamics. Among them, soybeans had the longest growth cycle, characterised by extended vegetative and early reproductive phases, while sugar peas developed the fastest. In all species, nitrate was the dominant form of nitrogen found in the roots, stems, and leaves, followed by ammonium (NH₃–N) and nitrite (NO₂⁻). The levels of NH₃–N varied among species, peaking early in faba beans and later in chickpeas. NO₂⁻ concentrations were low and decreased with development. The activities of nitrate reductase and nitrite reductase also varied across species, plant organs, and growth stages. The highest enzyme activity was consistently observed in the leaves. Notably, peas exhibited high enzyme activity across all organs, while the leaves of soybeans showed the highest activity in the studied legumes.

1. Introduction

Legumes are some of the most important agricultural crops, offering numerous benefits as a sustainable source of protein and bioactive compounds for both human and animal consumption. Legumes are effectively incorporated into various agricultural practices such as crop rotation, intercropping and green manure application. These issues have been extensively studied in field crop research and summarised in various scientific articles [1,2,3,4,5]. However, there is a significant knowledge gap regarding the cultivation of legumes in hydroponic systems. Soybeans are mainly grown and studied in hydroponic systems [6,7,8,9,10], but there is little data on the cultivation of other legumes such as sugar peas, lentils and others. Most of the studies have focused on the quality and productivity of these crops, examining different hydroponic systems, the effects of mineral nutrients and salinity, various Rhizobium strains, etc. [11,12,13,14,15]. Still, we have not found any data on nitrogen metabolism at different growth stages of legumes cultivated rhizobia-free in hydroponic solution with only mineral N. It is well known that legume growth in the field is a dynamic process closely linked to developmental stages and resource allocation. Initially, during germination, growth is minimal. In the vegetative stage, growth reaches its peak, characterised by rapid shoot and root development. As plants transition to reproductive development, growth slows until seed formation occurs [16,17,18,19]. Nitrogen uptake in legumes fluctuates significantly throughout their development. The process begins from seed germination to establishing symbiotic nitrogen fixation, and then to nitrogen remobilisation during reproduction. Nitrogen cycle enzyme activity peaks from the late vegetative stage to early flowering, when canopy growth and nitrogen demand are at their highest. During pod and seed development, their activity declines as nitrogen is redirected towards the reproductive systems [6,16,20,21,22,23]. However, there is limited understanding of how these processes occur when legumes are cultivated in hydroponic systems within controlled environment agriculture, where consistent environmental conditions are maintained. Therefore, our studies aimed to evaluate the growth of various legumes without rhizobia in hydroponics and to analyse ammonia, nitrite, and nitrate levels, as well as changes in nitrogen cycle enzyme activity during their growth. The results obtained can be used to screen legume species for future studies on intercropping with leafy vegetables, utilising the symbiosis between legumes and nitrogen-fixing bacteria. Such studies are currently lacking. According to the existing literature, research on hydroponic cultivation of both legumes and non-legumes has mainly focused on animal feed [24,25,26,27]. Notably, studies by D-Andrade et al. [28] on intercropping lettuce with alfalfa in a vertical hydroponic system demonstrated that combining moderate nitrate levels with nitrogen-fixing legumes improved resource use efficiency. This approach also reduced the need for synthetic fertilisers in hydroponic systems, without significantly impacting lettuce yield.

2. Materials and Methods

2.1. Plant Material and Growing Conditions

These legumes were grown: chickpea (Cicer arietinum, ‘Badil’), faba bean (Vicia faba, ‘Aquadulce’), lentil (Lens culinaris, beluga type), soybean (Glycine max, tofu-soya type, ‘Proteix’), and sugar pea (Pisum sativum, ‘Norli’—further in the text—pea). All legume seeds used in this study were purchased from Sativa Biosaatgut GmbH, Jestetten, Germany.

The pot experiment was conducted in a controlled environment within walk-in growth chambers (4 m × 6 m × 3.2 m) at the phytotron complex of the Institute of Horticulture, Research Centre for Agriculture and Forestry in Lithuania. The microclimate inside the growth chamber was autonomously controlled using the microclimate control system (2.5/3.4 kW, Panasonic Holdings Corporation, Tokyo, Japan).

Legume plant seeds were sown in rockwool cubes measuring 2.5 cm × 2.5 cm × 3.0 cm, which had been pre-soaked in deionised water adjusted to a pH of 5.0 using diluted sulfuric acid. These cubes were then placed in a plastic tray for germination. Seeds were disinfected using a 10% sodium hypochlorite solution for 10 min. They were then rinsed five times with sterile distilled water and soaked in distilled water for 2 h. However, soybean seeds were only disinfected and rinsed with sterile water, as they tend to swell quickly. Seeds were germinated in a growth chamber with an average temperature of 24 °C and a controlled relative humidity of 60 ± 5%. Artificial lighting was provided by 4-channel controllable light-emitting diode (LED) units (TUAS GTR 2V 0021096109 C1 DL ST, Tungsram, Budapest, Hungary). The photosynthetic photon flux density (PPFD) was maintained at 200 ± 10 μmol m⁻² s⁻¹ throughout the 16 h photoperiod, with a preselected spectral composition of deep red (61%), blue (20%), white (15%), and far red (4%). PPFD was measured and regulated at the plant level using a photometer-radiometer (RF-100, Sonopan, Bialystok, Poland).

After 10 days, the plants that had developed their first true leaves were transferred to 35 L deep-water culture (DWC) hydroponic systems comprising three independent tanks filled with nutrient solution. One tank represents one experimental replication. Three random plants from each replicate were measured. The mean of the three plants was used as one value per replicate for statistical analysis. The solution contained the following concentrations (mg L⁻¹): nitrogen 120 (109.14 mg L⁻¹ NO₃⁻–N and 10.86 mg L⁻¹ NH₄⁺–N in the final solution), calcium 88, phosphorus 20, potassium 128, magnesium 40, sulphur 53, boron 0.16, molybdenum 0.2, manganese 0.08, copper 0.08, iron 1.6, and zinc 0.8. The pH of the solution was maintained between 6.0 and 6.5, and the electrical conductivity (EC) was set at 1.2 mS cm⁻¹. A portable meter (GroLine HI9814, Hanna Instruments, Woonsocket, RI, USA) was used to measure the pH and EC of the hydroponic solution daily, with adjustments made using sulfuric acid or potassium hydroxide as necessary. The hydroponic solution was saturated with oxygen using air pumps (ProSilent a400 JBL GmbH & Co. KG, Neuhofen, Germany) capacity 600 L h⁻¹, pressure > 160 mbar). The hydroponic systems were replenished with 7 L of the nutrient solution (same as mentioned above) for chickpeas and peas, 10 L for faba beans and 5 L for lentils, every week from the R2 (see Section 2.2) growth stage until the end of the experiment. For soybeans, hydroponic systems were replenished weekly with 5 L of solution from the V4 (see Section 2.2) growth stage to R2, and with 10 L from R2 until the end of the experiment. Plants were grown at an average day/night temperature of 23/19 °C.

2.2. Growth Characteristics and Sampling

Three randomly selected plants from each replication at various growth stages (Vn—the legume plant develops new leaves, R2—full bloom, then most of the flowers on the plant are open, R3—early pod, then the first pods become visible) were selected for growth indices measurements and biochemical analysis. Further, only the development of legumes was monitored, and growth stages R4 (full pod; pods reach their full size but are still flat) and R5 (early seeds were recorded) were monitored. Growth stages were evaluated according to the relevant agricultural manuals [29,30,31]. The growth characteristics of legumes were studied by measuring various parameters, including plant height, root length, and the fresh weights (FW) and dry weights (DW) of leaves, stems, and roots. FW and DW were measured using an electronic balance (Mettler Toledo, ML104T/00; Mettler-Toledo, Columbus, OH, USA). DW was determined by drying the samples for 48 h in a drying oven (Venticell-222, Medcenter Einrichtungen, Gräfeling, Germany) at 70 °C. The following indices were calculated based on the data from DW [32,33]:

Relative growth rate (RGR)

$$\mathrm{RGR}={\displaystyle \frac{\mathrm{l}\mathrm{n}\left({\mathrm{W}}_2\right)-\mathrm{l}\mathrm{n}\left({\mathrm{W}}_1\right)}{{\mathrm{t}}_2-{\mathrm{t}}_1}}$$

(1)

where W₁ and W₂ are the plant dry weights at times t₁ and t₂, respectively; ln—denotes the natural logarithm; t₂ − t₁ is the time interval over which growth is measured. The resulting RGR is typically expressed in units such as g g⁻¹ day⁻¹.

Leaf weight ratio (LWR)

$$\mathrm{L}\mathrm{W}\mathrm{R}={\displaystyle \frac{\mathrm{Dry} \mathrm{weight} \mathrm{of} \mathrm{leaves}}{\mathrm{Total} \mathrm{dry} \mathrm{weight} \mathrm{of} \mathrm{the} \mathrm{plant}}}$$

(2)

Root to shoot ratio (RSR)

$$\mathrm{R}\mathrm{S}\mathrm{R}={\displaystyle \frac{\mathrm{Dry} \mathrm{weight} \mathrm{of} \mathrm{roots}}{\mathrm{Dry} \mathrm{weight} \mathrm{of} \mathrm{shoots} }}$$

(3)

The dry weight of legumes was used to determine ammonia, nitrites, and nitrates, while the fresh weight was used to assess nitrite and nitrate reductase activity.

2.3. Determination of Ammonia, Nitrites, and Nitrates

Dried plant material (approximately 0.5 g) was weighed into a 50 mL disposable centrifuge tube and extracted with 50 mL of hot (95 °C) water for 60 min using a test tube rotator Labinco LD79 (Labinco BV, Breda, The Netherlands). After extraction samples were centrifuged at 3000 rpm for 30 min.

Determination of nitrogen compounds was performed using Hanna Instruments HI83306 multiparameter photometer (Hanna Instruments, Woonsocket, RI, USA). Ammonia was determined as NH₃–N using an adapted Nessler method, measuring absorbance at 420 nm. 1 mL of unreacted sample is added to the cuvette using a 1 mL pipette. 9 mL of ammonia High Range Reagent B is added. The plastic stopper and the cap are replaced, and the cuvette is swirled to mix the solution. Zero reading is taken. After that, 4 drops of ammonia reagent A are added, the stopper and cap are replaced, the cuvette is swirled and reinserted to photometer, the timer is switched on, and the measurement is taken after 3.5 min. The result is displayed as mg/L NH₃–N (Method Adaptation of the ASTM Manual of Water and Environmental Technology), D1426, Nessler Method [34].

Nitrites were determined using the method Adaptation of the Ferrous Sulfate Method. A light source LED with a narrow-band interference filter at 575 nm was used. The cuvette is filled with 10 mL of unreacted sample, and the plastic stopper and the cap are replaced. The cuvette is inserted into the holder, and a zero measurement is taken. One packet of Nitrite High Range Reagent is added to the solution, and the plastic stopper and the cap are replaced. The cuvette is gently shaken until the reagent is completely dissolved (about 2 min). After insertion of the cuvette into the holder, the timer is pressed (10 min), and after that, the reading is performed. The instrument displays nitrite (NO₂⁻) concentration in mg L⁻¹.

Nitrates were determined using an adaptation of the cadmium reduction method. A light source LED with a narrow band interference filter @ 525 nm was used. The cuvette is filled with 0.2 mL of sample, and 9.8 mL of ultra-pure water is added to bring the sample volume to 10 mL. The plastic stopper and cap are replaced. The cuvette is inserted into the holder, and the blank is measured. One packet of Nitrate reagent is added to the solution, the plastic stopper and the cap are replaced, and the contents of the cuvette are vigorously shaken up and down for exactly 10 s. Then the mixing is continued by gently inverting the cuvette for 50 s, taking care not to introduce air bubbles. After that, the cuvette is reintroduced to the photometer, the timer is started, and after 4.5 min, the colour development is measured; the instrument displays the results in mg L⁻¹ of nitrate-nitrogen (NO₃⁻).

2.4. Determination of Nitrite and Nitrate Reductase Activity

Fresh plant material samples were ground in liquid nitrogen and homogenised in extraction buffer (100 mM potassium phosphate, pH 7.5; 5 mM magnesium acetate; 10% (v/v) glycerol; 10% (w/v) PVPP; 0.1% (v/v) Triton X-100; 1 mM EDTA; 1 mM DTT; 1 mM PMSF; 1 mM benzamidine; 1 mM 6-aminocaproic acid). The homogenate was filtered and centrifuged at 14,000 rpm for 15 min, and the supernatant was used for enzyme activity analysis immediately after preparation.

Nitrate reductase (NR) activity was determined by measuring the formation of nitrite (NO₂⁻) in an NADH-dependent reaction. The reaction mixture consisted of 13 µL of enzyme extract and 67 µL of the analytical solution (50 mM potassium phosphate, pH 7.5; 5 mM EDTA; 0.25 mM NADH; 5 mM KNO₃), incubated for 20 min at 25 °C. The reaction was stopped by adding 0.12 mL of a 1:1 (v/v) mixture of sulfanilamide (1% (w/v) 3 M HCl) and NED (0.1% (w/v)), then the samples were incubated for 15 min at 25 °C, and the absorbance at 540 nm was measured (BMG Labtech microplate reader, Ortenberg, Germany) [35]. A similar analytical solution without NADH was used as a blank. The amount of NO₂⁻ was calculated from the NaNO₂ calibration curve, and the NR activity was expressed as nmol NO₂⁻ g⁻¹ FW h⁻¹.

Nitrite reductase (NiR) activity was determined by nitrite (NO₂⁻) consumption in a methyl viologen/dithionite reducing system [35]. The reaction mixture consisted of 140 µL of 100 mM potassium phosphate buffer (pH 7.5), 10 µL of 5 mM KNO₂, 10 µL of enzyme extract, 10 µL of methyl viologen (2 mg mL⁻¹) and 10 µL of deionised water; the reaction was initiated by the addition of 20 µL of sodium dithionite (25 mg mL⁻¹) in 290 mM NaHCO₃ solution and incubated for 30 min at 25 °C.

After incubation, 10 µL of the reaction mixture was diluted with 190 µL of deionised water (to oxidise dithionite), then 0.12 mL of a 1:1 (v/v) mixture of sulfanilamide (1% (w/v) 3 N HCl) and NED (0.05% (w/v)) 0.06 mL of sulfanilamide (1% (w/v) 3 M HCl) were added, incubated for 30 min at 25 °C and the absorbance at 540 nm was measured (BMG Labtech microplate reader, Ortenberg, Germany). The mixture without methyl viologen was used as a blank. The amount of consumed (bound) NO₂⁻ was estimated from the nitrite calibration curve, and the NiR activity was expressed as mmol NO₂⁻ g⁻¹ FW h⁻¹.

2.5. Statistical Analysis

A statistical analysis was performed using Microsoft Excel 2016 and XLSTAT 2022 (Addinsoft, Long Island, NY, USA). A one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference test (p < 0.05) for multiple comparisons was used to evaluate differences in mean measurements (n = 3).

3. Results

3.1. The Growth and Development of Legumes in Hydroponics

The duration of the developmental stages varies among the five legume species (Figure 1). In all crops, the early vegetative (VE–Vn) and reproductive (till full bloom (R2)) stages occupy a large portion of total development, particularly in soybean and faba bean. Before transitioning to reproductive stages, the vegetative growth stage (VE–Vn) of soybean lasted 49 days, whereas that of faba bean lasted 33 days. Soybeans took the longest to grow, reaching only the R4 growth stage within 97 days of cultivation, indicating that full pod formation had occurred. Given that this long-growing plant did not align with our subsequent goals of co-cultivating legumes with leafy vegetables, soybeans were not grown beyond the R5 growth stage. The shortest vegetative growing period was observed in sugar pea, lasting just 22 days. During reproductive development, the duration of individual stages in sugar pea was similar to that in lentil and chickpea. Overall, their growth to the R5 stage lasted an average of about 60 days.

Morphological traits and biomass accumulation were evaluated at V2, R2 and R3 growth stages and showed at

Table 1. Changes in growth parameters during the growth of legume plants.

Indices	GS	Legumes Plants
		Chickpea	Faba Beans	Lentil	Soybean	Sugar Pea
Shoot height, cm	V2	16.50 ± 1.500 c	21.00 ± 1.732 b	33.67 ± 0.294 c	32.73 ± 0.623 b	13.67 ± 0.333 c
	R2	39.00 ± 2.887 b	58.50 ± 1.443 a	50.33 ± 0.588 b	83.67 ± 1.856 a	25.67 ± 0.667 b
	R3	55.67 ± 3.283 a	64.00 ± 3.000 a	61.00 ± 1.018 a	91.00 ± 5.196 a	32.67 ± 1.202 a
Root length, cm	V2	39.33 ± 2.848 b	35.50 ± 0.289 c	31.67 ± 0.401 b	73.00 ± 6.245 a	47.67 ± 5.897 a
	R2	58.33 ± 2.963 a	58.67 ± 4.485 b	56.33 ± 0.111 a	63.00 ± 3.786 a	57.67 ± 2.963 a
	R3	64.00 ± 1.732 a	92.50 ± 6.640 a	53.67 ± 0.801 a	78.00 ± 2.309 a	56.67 ± 6.360 a
Leaf DW, g	V2	0.15 ± 0.016 b	0.66 ± 0.054 b	0.23 ± 0.009 b	1.18 ± 0.088 b	0.48 ± 0.059 c
	R2	2.04 ± 0.383 b	6.52 ± 0.547 a	1.28 ± 0.020 a	11.11 ± 2.226 b	1.49 ± 0.081 b
	R3	6.77 ± 1.014 a	7.84 ± 1.110 a	1.38 ± 0.046 a	35.36 ± 8.233 a	2.08 ± 0.071 a
Stem DW, g	V2	0.04 ± 0.007 b	0.30 ± 0.033 b	0.14 ± 0.006 b	0.44 ± 0.024 b	0.19 ± 0.030 c
	R2	0.87 ± 0.184 b	4.84 ± 0.533 a	1.09 ± 0.006 a	7.10 ± 1.048 b	1.09 ± 0.056 b
	R3	3.92 ± 0.317 a	6.32 ± 0.919 a	1.49 ± 0.199 a	23.26 ± 6.027 a	1.89 ± 0.112 a
Root DW, g	V2	0.05 ± 0.008 c	0.31 ± 0.025 c	0.11 ± 0.007 b	0.36 ± 0.026 b	0.16 ± 0.022 c
	R2	2.08 ± 0.307 b	2.62 ± 0.223 b	0.48 ± 0.026 a	1.37 ± 0.126 b	0.42 ± 0.018 b
	R3	3.07 ± 0.126 a	5.49 ± 0.837 a	0.58 ± 0.057 a	13.95 ± 1.859 a	0.66 ± 0.037 a
Total DW, g	V2	0.24 ± 0.029 c	1.26 ± 0.112 b	0.48 ± 0.021 b	1.98 ± 0.126 b	0.84 ± 0.109 c
	R2	4.98 ± 0.849 b	13.98 ± 1.303 a	2.85 ± 0.040 a	19.58 ± 3.106 b	2.99 ± 0.142 b
	R3	13.76 ± 1.214 a	19.64 ± 2.124 a	3.45 ± 0.413 a	72.57± 16.118 a	4.64 ± 0.162 a
RGR	VE–V2	0.04 ± 0.001 c	0.04 ± 0.001 b	0.26 ± 0.002 a	0.14 ± 0.001 a	0.13 ± 0.004 a
	V2–R2	0.16 ± 0.002 a	0.11 ± 0.001 a	0.08 ± 0.001 b	0.06 ± 0.002 b	0.08 ± 0.022 ab
	R2–R3	0.11 ± 0.010 b	0.02 ± 0.001 c	0.03 ± 0.014 c	0.06 ± 0.005 b	0.04 ± 0.002 b
LWR	V2	0.61 ± 0.010 a	0.52 ± 0.003 a	0.49 ± 0.004 a	0.60 ± 0.009 a	0.58 ± 0.004 a
	R2	0.41 ± 0.016 b	0.47 ± 0.004 ab	0.45 ± 0.006 ab	0.56 ± 0.023 a	0.50 ± 0.004 b
	R3	0.49 ± 0.032 b	0.40 ± 0.032 b	0.40 ± 0.022 b	0.48 ± 0.006 a	0.45 ± 0.002 c
RSR	V2	0.27 ± 0.027 b	0.33 ± 0.004 a	0.30 ± 0.007 a	0.22 ± 0.016 a	0.24 ± 0.015 a
	R2	0.74 ± 0.086 a	0.23 ± 0.002 a	0.21 ± 0.010 a	0.08 ± 0.020 a	0.16 ± 0.009 b
	R3	0.30 ± 0.048 b	0.40 ± 0.078 a	0.21 ± 0.047 a	0.20 ± 0.021 a	0.17 ± 0.015 b

Notes: GS—growth stages, VE—germination and emergence, V2—the legume plant leaves, R2—full bloom, R3—early pod; RGR—relative growth rate, LWR—leaf weight ratio, RSR—root to shoot ratio. The values are means ± SE (standard error). According to Tukey’s test, different letters in columns indicate significant differences at p < 0.05 level.

. As development progressed, signs of senescence, such as stunted growth and yellowing leaves, became evident, so these measurements were not conducted at R4 and R5 growth stages. Shoot height increased steadily from V2 to R3 in all species. At the V2 stage, lentil and soybean exhibited the greatest shoot height, followed by faba bean, whereas chickpea and sugar pea were significantly shorter. These trends remained evident throughout the growth period.

Root length also varied significantly, but the extent of this variation depended on the legume species. Soybeans produced long roots consistently at all stages, with no significant differences between vegetative and reproductive stages. Similar trends have also been observed in peas. The roots of the faba bean grew consistently longer, with R3 exhibiting the greatest length, significantly surpassing those of other varieties. The root length of chickpeas and lentils was shorter during the vegetative stage than during the reproductive stage; however, no significant differences were observed among the various reproductive stages.

Biomass accumulation increased significantly from V2 to R3 in all five legume species, with substantial differences among organs and crops. At V2 and R2, soybean and faba bean exhibited the greatest leaf, stem, and root dry weights. However, at R3, only soybeans showed the highest growth across all organs, resulting in a total dry weight of 72.57 g, far exceeding that of all other species. Chickpea, sugar pea and lentil produced comparatively little biomass. The biomass changes in legume roots differed from those in the shoots. Root dry weight increased at V2 and R2, with chickpea and faba bean exhibiting the highest values. Faba bean and chickpea also increased substantially at R3, although to a lesser extent compared to soybeans, whereas lentil and sugar pea consistently produced the lowest root biomass across stages. Overall, there was significant variation in biomass partitioning and total dry matter accumulation among species, with soybean demonstrating the most vigorous growth during vegetative and reproductive development, unlike sugar pea and lentil.

Growth indices also differed significantly among species. Lentil exhibited the highest RGR during VE–V2 and decreased as the plants developed. Similar trends have been observed in both soybeans and sugar peas. Meanwhile, chickpea and faba bean showed the greatest RGR from V2–R2. LWR generally declined with growth in all species except soybean, which maintained consistently high values. Chickpeas showed a significant increase in RSR at R2, unlike other legumes, which did not exhibit substantial differences in this index during their development. The only exception was sugar peas, where the RSR was higher at V2. RSR in soybeans showed consistently low values across various growth stages due to its predominant shoot biomass accumulation. Overall, these findings reveal significant interspecific differences in growth behaviour, biomass distribution, and developmental pathways among legume species.

3.2. Nitrogen Content in Legumes

Significant differences in the concentrations of NH₃–N, NO₂⁻, and NO₃⁻ were observed in the roots, stems, and leaves of various legume species at different growth stages (

Table 2. Changes in ammonia nitrogen, nitrites, and nitrates content during the growth of legume plants.

Indices	GS	Legumes Plants
		Chickpea	Faba Beans	Lentil	Soybean	Sugar Pea
Roots
NH3–N, mg g−1 DW	V2	0.23 ± 0.009 c	16.85 ± 0.029 a	0.80 ± 0.026 a	0.45 ± 0.028 c	0.57 ± 0.009 b
	R2	1.68 ± 0.017 b	11.17 ± 0.521 b	0.54 ± 0.022 c	0.77 ± 0.021 b	0.46 ± 0.007 c
	R3	2.02 ± 0.046 a	4.93 ± 0.109 c	0.71 ± 0.007 b	2.96 ± 0.015 a	0.67 ± 0.038 a
NO2−, mg g−1 DW	V2	0.80 ± 0.001 c	0.87 ± 0.088 b	2.37 ± 0.133 a	1.43 ± 0.088 a	1.33 ± 0.088 a
	R2	1.10 ± 0.058 b	0.27 ± 0.033 c	2.03 ± 0.067 a	1.10 ± 0.115 a	0.97 ± 0.033 a
	R3	1.37 ± 0.067 a	1.23 ± 0.088 a	1.90 ± 0.153 a	1.67 ± 0.517 a	0.83 ± 0.233 a
NO3−, mg g−1 DW	V2	52.67 ± 4.419 c	79.17 ± 1.093 b	107.50 ± 2.291 a	100.17 ± 0.726 a	74.50 ± 2.843 b
	R2	115.00 ± 4.907 a	89.33 ± 2.455 a	115.67 ± 3.678 a	107.83 ± 4.807 a	84.33 ± 2.186 ab
	R3	91.00 ± 2.000 b	80.17 ± 1.093 b	103.50 ± 3.547 a	104.83 ± 1.590 a	86.50 ± 2.000 a
Stems
NH3–N, mg g−1 DW	V2	0.00 ± 0.000 c	11.56 ± 0.161 a	1.17 ± 0.013 b	0.76 ± 0.037 a	2.18 ± 0.006 c
	R2	0.33 ± 0.001 a	7.81 ± 0.223 b	0.76 ± 0.010 c	0.74 ± 0.007 a	2.86 ± 0.071 a
	R3	0.27 ± 0.012 b	4.67 ± 0.048 c	1.25 ± 0.011 a	0.56 ± 0.012 b	2.38 ± 0.012 b
NO2−, mg g−1 DW	V2	3.95 ± 0.180 a	2.66 ± 0.067 a	1.16 ± 0.066 a	2.23 ± 0.033 a	1.38 ± 0.037 a
	R2	0.60 ± 0.099 b	1.43 ± 0.066 b	0.36 ± 0.033 b	1.96 ± 0.218 ab	1.07 ± 0.033 b
	R3	0.23 ± 0.033 b	1.01 ± 0.065 c	0.33 ± 0.033 b	1.56 ± 0.033 b	0.99 ± 0.057 b
NO3−, mg g−1 DW	V2	95.49 ± 1.197 a	63.41 ± 1.945 a	82.59 ± 1.477 a	61.96 ± 1.727 a	80.25 ± 1.659 a
	R2	73.23 ± 1.906 b	52.51 ± 0.879 b	68.68 ± 0.760 b	60.94 ± 1.257 a	78.79 ± 0.333 a
	R3	65.89 ± 1.900 b	56.30 ± 0.866 b	51.36 ± 1.482 c	54.97 ± 2.711 a	67.39 ± 1.876 b
Leaves
NH3–N, mg g−1 DW	V2	0.16 ± 0.079 c	13.88 ± 0.217 a	1.13 ± 0.044 a	1.16 ± 0.036 a	1.00 ± 0.029 b
	R2	0.45 ± 0.001 b	8.37 ± 0.192 b	1.38 ± 0.044 a	1.27 ± 0.015 a	1.03 ± 0.014 b
	R3	0.81 ± 0.030 a	4.33 ± 0.176 c	1.27 ± 0.145 a	1.26 ± 0.030 a	1.53 ± 0.014 a
NO2−, mg g−1 DW	V2	2.10 ± 0.058 b	3.63 ± 0.067 b	2.87 ± 0.033 a	4.13 ± 0.935 a	2.17 ± 0.067 c
	R2	3.73 ± 0.167 a	3.93 ± 0.033 a	2.13 ± 0.120 b	2.63 ± 0.033 a	2.63 ± 0.067 b
	R3	3.10 ± 0.300 a	3.37 ± 0.033 c	2.47 ± 0.088 b	3.17 ± 0.318 a	2.93 ± 0.033 a
NO3−, mg g−1 DW	V2	46.67 ± 3.087 a	58.17 ± 1.590 a	63.83 ± 2.920 a	45.33 ± 3.245 a	68.67 ± 1.014 a
	R2	54.67 ± 3.383 a	55.17 ± 3.528 a	63.83 ± 1.878 a	55.33 ± 4.842 a	63.67 ± 3.444 a
	R3	50.67 ± 4.919 a	51.33 ± 3.346 a	54.83 ± 1.093 a	48.33 ± 2.603 a	60.17 ± 3.180 a

Notes: GS—growth stages, V2—the legume plant leaves, R2—full bloom, R3—early pod; NH₃–N—ammonia nitrogen, NO₂⁻—nitrites, NO₃⁻—nitrates; DW—dry weight. The values are means ± SE (standard error). According to Tukey’s test, different letters in columns indicate significant differences at p < 0.05 level.

). At the V2 stage, faba bean exhibited markedly higher NH₃–N levels compared to all other species. However, this concentration decreased significantly as the faba bean developed. In contrast, the roots of chickpea and soybean showed a progressive increase in NH₃–N from the V2 to the R3 growth stages, with significant differences noted. Lentil and pea maintained relatively low concentrations of NH₃–N across all growth stages, with the lowest levels recorded at the R2 stage. Lentil also exhibited the highest levels of NO₂⁻ and NO₃⁻ in its roots throughout all growth stages, with no significant changes noted during development. While chickpea showed significantly lower levels of NO₂⁻, these increased considerably from the V2 to R3 growth stages. No significant variations in NO₂⁻ concentrations were observed among the growth stages of soybean and pea.

Chickpea recorded the lowest NO₃⁻ concentration at the V2 stage, but this increased as the plant developed, particularly at the R2 growth stage. A similar trend was observed in faba beans. However, at the R3 growth stage, nitrate concentrations slightly declined in the roots of both chickpea and faba bean. Pea maintained moderate nitrate levels, with a slight increase across the stages.

The stems of chickpea and pea showed the highest accumulation of NH₃–N at the R2 growth stage, compared to the V2 and R3 stages. In contrast, lentil stems exhibited an opposite trend. The highest concentration of NO₂⁻ in chickpea stems was observed at the V2 growth stage, followed by faba bean and soybean. At later growth stages, soybean maintained the highest NO₂⁻ levels among the legumes studied. Notably, at the V2 growth stage, chickpea displayed a higher concentration of NO₃⁻ in comparison with other legume species. Throughout development, the levels of NO₂⁻ and NO₃⁻ decreased in the stems of all species, with chickpea experiencing the most pronounced decline. At the R2 and R3 growth stages, pea exhibited the highest NO₃⁻ concentrations compared to the other legumes.

At the V2 growth stage, chickpea leaves exhibited the lowest NH₃–N concentration, which progressively increased from V2 to R3, reaching a peak at R3. A similar trend was observed in peas. In contrast, the NH₃–N concentrations in lentils and soybeans showed no significant changes throughout their growth stages. At V2, soybeans had the highest concentration of NO₂⁻ in their leaves, although growth stages did not significantly affect this measurement. During the R2 and R3 stages, the NO₂⁻ concentration was significantly highest in chickpeas and faba beans, while lentils showed the lowest levels. Additionally, leaf NO₃⁻ concentrations did not differ significantly among the legume species at any growth stage.

The results indicated that, across all species and growth stages, NO₃⁻ was the most abundant form of nitrogen found in the roots, leaves, and stems of the studied legumes. This was followed by NH₃–N and NO₂⁻. The levels of NH₃–N showed significant variation among species, particularly in faba bean and chickpea. In faba bean, peak concentrations of NH₃–N occurred during the early growth stages, whereas in chickpea, they were observed at later stages. NO₂⁻ concentrations were generally lower and decreased as plants developed, especially in stems. These patterns suggest that species and tissue type strongly regulate the accumulation of different nitrogen forms during legume growth.

3.3. Nitrite and Nitrate Reductase Activity in Legumes

Root NR and NiR activities varied significantly across species and growth stages. Pea plants exhibited the highest activity of these enzymes in their roots during the V2 growth stage. As they progressed into reproductive development, the activity of these enzymes decreased, but it remained higher than that observed in other plants. Additionally, no significant differences were noted between the R2 and R3 growth stages. In contrast, faba beans experienced a substantial decrease in both NR and NiR activity as they developed. Lentils at the V2 growth stage showed high NR activity in roots, but NiR activity was lower than in other legumes (

Table 3. Changes in nitrite and nitrate reductase activity during the growth of legume plants.

Indices	GS	Legumes Plants
		Chickpea	Faba Beans	Lentil	Soybean	Sugar Pea
Roots
NR, μmol g−1 FW h−1	V2	18.93 ± 0.799 ab	46.05 ± 0.782 a	67.92 ± 0.015 a	37.08 ± 0.612 a	70.81 ± 0.356 a
	R2	21.41 ± 0.702 a	35.63 ± 2.818 b	19.07 ± 0.173 c	22.60 ± 0.958 b	54.63 ± 1.287 b
	R3	16.46 ± 1.299 b	21.41 ± 0.702 c	25.36 ± 0.941 b	23.63 ± 0.836 b	58.48 ± 1.236 b
NiR, μmol g−1 FW h−1	V2	24.75 ± 0.835 a	37.75 ± 1.790 a	18.88 ± 0.447 a	35.19 ± 0.587 a	50.58 ± 1.051 a
	R2	24.42 ± 1.962 a	35.19 ± 0.587 a	25.50 ± 0.964 a	38.89 ± 1.504 a	51.07 ± 0.977 a
	R3	25.09 ± 0.725 a	24.42 ± 1.962 b	22.29 ± 3.438 a	25.52 ± 2.849 b	50.61 ± 1.068 a
Stems
NR, μmol g−1 FW h−1	V2	54.55 ± 1.634 b	52.66 ± 0.211 b	84.36 ± 0.222 a	59.96 ± 2.211 a	121.59 ± 0.872 a
	R2	67.92 ± 1.859 a	69.59 ± 2.119 a	65.36 ± 5.887 b	65.94 ± 2.996 a	72.63 ± 0.938 b
	R3	41.18 ± 1.997 c	67.92 ± 1.859 a	86.07 ± 4.316 a	39.86 ± 1.212 b	71.77 ± 2.165 b
NiR, μmol g−1 FW h−1	V2	54.27 ± 1.708 a	53.37 ± 0.309 a	52.36 ± 0.294 a	47.72 ± 5.468 a	60.79 ± 0.436 b
	R2	56.02 ± 1.839 a	47.72 ± 5.468 a	52.78 ± 1.970 a	27.18 ± 1.019 b	66.78 ± 0.121 a
	R3	52.51 ± 2.212 a	56.02 ± 1.839 a	52.36 ± 0.294 a	34.01 ± 5.387 ab	53.04 ± 1.279 c
Leaves
NR, μmol g−1 FW h−1	V2	290.92 ± 4.323 a	329.63 ± 1.617 b	307.87 ± 9.739 a	587.34 ± 2.584 a	507.07 ± 7.853 a
	R2	284.18 ± 9.674 a	377.80 ± 2.607 a	371.82 ± 28.936 a	563.46 ± 26.142 a	411.88 ± 41.266 ab
	R3	297.66 ± 9.601 a	284.18 ± 9.674 c	371.48 ± 8.324 a	473.69 ± 8.625 b	337.11 ± 17.39 b
NiR, μmol g−1 FW h−1	V2	310.93 ± 5.340 ab	341.83 ± 11.621 a	156.58 ± 1.943 b	325.41 ± 16.887 b	328.50 ± 13.888 a
	R2	341.83 ± 11.621 a	325.41 ± 16.887 a	294.21 ± 10.776 a	445.28 ± 10.290 a	320.66 ± 5.633 a
	R3	280.03 ± 2.265 b	341.83 ± 11.621 a	155.04 ± 1.755 b	443.62 ± 15.516 a	337.85 ± 8.487 a

Notes: GS—growth stages, V2—the legume plant leaves, R2—full bloom, R3—early pod. NR—nitrate reductase, NiR—nitrite reductase. The values are means ± SE (standard error). According to Tukey’s test, different letters in columns indicate significant differences at p < 0.05 level.

).

Similar trends in NR and NiR activity changes during development were observed in pea stems, mirroring those in roots. In chickpea and soybean stems, NR activity decreased, particularly at the R3 growth stage. In contrast, lentil stems exhibited relatively high NR activity, especially during the V2 and R3 growth stages. However, the NiR activity in the stems of these plants remained consistent across all growth stages. Analogous trends were also observed in both chickpea and faba bean stems.

Leaves showed significantly higher activities of both NR and NiR compared to roots and stems across all species and growth stages. In particular, NR activity was notably elevated in soybean and pea leaves across all growth stages, with a peak at the V2 stage. For chickpea and lentil leaves, NR activity remained consistent across all growth stages. Additionally, NiR activity in soybean leaves increased during the reproductive development stage compared to the vegetative stage, and it was higher than that in other leguminous plants. In contrast, NiR activity in faba bean and pea leaves did not vary with growth stage.

In summary, the activities of NR and NiR varied significantly across legume species, plant organs, and growth stages. The leaves of all legume plants displayed much higher NR and NiR activity than their roots and stems. Among the legumes studied, peas showed high enzyme activity across all plant parts. Notably, soybean leaves demonstrated the highest levels of NR and NiR activity compared to the other legumes.

4. Discussion

4.1. Characteristics of Growth and Development of Legumes in Hydroponics

The development of legumes in hydroponic systems CEA is influenced by the interaction between the genetic traits of specific species and carefully controlled environmental conditions. CEA systems enable precise regulation of temperature, light intensity, photoperiod, nutrient composition, root-zone oxygenation, and water availability across various plant cultivation systems. All these factors also significantly impact legume phenology, growth rates, and biomass [10,36,37,38]. Our results showed that variation in developmental stage duration among the five legume species highlights well-established species-specific differences in phenological progression and growth strategies under hydroponic cultivation in CEA. Similar trends to our results have been observed in other studies in which legume plants were grown in hydroponic systems under various conditions [10,39,40]. Soybeans and peas were the most notable in terms of their development duration. Soybeans exhibit longer early vegetative (VE–Vn) and early reproductive (up to full bloom, R2) stages compared to other legumes, indicating a prolonged period for canopy establishment and resource accumulation. The literature shows that soybeans are primarily known for their sensitivity to photoperiod and for regulating vegetative growth. The length of the vegetative phase significantly affects the timing of flowering and maturation, particularly under short-day conditions, and is influenced by significant maturity loci and variations in genes associated with flowering time. These genetic and physiological factors contribute to the extended vegetative phases observed in many soybean genotypes, especially in long-day environments [17,41]. Generally, field experiments on soybean cultivars indicate that the average vegetative growth stage is about 40–65 days [17,42]. In our studies, soybean vegetative growth in hydroponic CEA lasted 49 days and was comparable to that in the field. This similarity may have been influenced by the long-day (16 h) environments under which the plants were cultivated. The relatively long vegetative growth stage led to an extended reproductive development phase. It took 97 days for the soybean to reach the R4 growth stage. In comparison, most soybean cultivars achieve physiological maturity (R7–R8) in about 100 to 130 days [43]. This indicates that further studies are necessary to assess the impact of photoperiod on soybean growth in hydroponic CEA.

Our results revealed that pea exhibited the shortest overall growth duration, taking just 60 days to reach the R5 stage, with 22 days in the vegetative growth stage. Literature data showed that the beginning of seed growth occurred in field pea at 75 days after sowing [21]. Phenological models developed for field-grown peas indicate that temperature and thermal time accumulation significantly influence the rate of development across the BBCH-coded stages. Quick transitions from vegetative to reproductive stages in peas have been modelled and observed, particularly under spring sowing and higher temperatures, aligning with the rapid onset of early reproduction in short-season systems [44]. During our research, we found that the average day/night temperature for growing peas hydroponically was 23 °C during the day and 19 °C at night. This may have contributed to their faster development compared to other legumes.

For our research, we cultivated legumes until the R3 growth stage, when pod formation began and, according to the literature, nitrogen fixation began to decline. In addition, maximum nitrogen fixation typically occurs during the late vegetative to early reproductive stages [21,45,46]. In the literature, it is noted that legumes focus on developing their canopy and root systems during the vegetative phase. This early growth phase emphasizes rapid leaf and stem expansion, which enhances photosynthesis, increases biomass accumulation, and supports the formation of early nodules. Once the plants begin flowering and forming pods, they enter reproductive growth, which involves boosting overall biomass and reallocating carbon to the reproductive organs [16,17,18,19,45,47]. Our studies indicate that hydroponically grown legumes exhibit growth patterns similar to those grown in the field, with increased biomass accumulation during the early pod growth stage (R3). Only chickpeas and peas grew reliably from the R2 to R3 growth stages. The mass of other legumes increased but did not differ significantly between these stages. Among all the legumes, soybeans were the most notable. Soybeans exhibited vigorous growth, with strong shoot and root development. These plants grown in the hydroponic system exhibited uneven growth, as shown by the lack of significant statistical differences in biomass between the V2 and R2 growth stages. This suggests that vigorous growth and high plant density in the hydroponic environment may have led to intraspecific competition among soybeans. There is currently no literature on how density affects the growth of individual soybeans in hydroponics. However, studies conducted in soil and open hydroponics indicate that increased density can reduce the number of branches on soybean plants [48,49].

Plant growth analysis is a valuable set of quantitative methods used to describe and interpret the performance of all plant systems, whether they are grown in natural or controlled conditions. This approach integrates various aspects of plant form and function, utilising simple data such as plant weight, area, volume, and composition. By analysing this data, we can investigate processes across the entire plant or crop [32,33]. Generally, RGR is highest during early vegetative growth, characterised by rapid leaf development, high rates of photosynthesis, and efficient biomass accumulation [32,37]. Our research has revealed differences in relative growth rates (RGR) among various legume species. Lentil, soybean, and pea growth rates (RGR) were highest during the early stages of development (VE–V2). Chickpea and faba bean reached their maximum RGR later (V2–R2), indicating a prolonged vegetative growth phase before they began reproduction. LWR measures the proportion of total plant biomass attributed to leaves during different stages of growth. This ratio is highest early in vegetative development, as leaves optimise light capture and carbon assimilation [32]. Our results supported these trends observed in the hydroponic growth of legumes. In faba bean, lentil, and soybean, this index remained largely unchanged during the transition to reproductive development, except in pea. Similar trends were observed when evaluating RSR, an important parameter for measuring the relative distribution of biomass between root and aboveground components [32]. Chickpea distinguished itself by having the highest RSR and the lowest LWR at the R2 stage, indicating increased rooting during early reproductive growth. Similar patterns have been observed when these plants are grown in the field [50].

In summary, distinct differences were observed in growth duration, phenology, and biomass distribution among the legumes studied, which aligns with field cultivation patterns. Soybean exhibited the longest growth cycle, characterised by extended vegetative and early reproductive stages. In contrast, pea developed the fastest, likely due to favourable temperature conditions that hastened its transition to reproduction. Given these developmental characteristics, pea may be the most suitable choice for identifying plants that can be cultivated alongside leafy vegetables. However, further research is needed.

4.2. Changes in Nitrogen Metabolism in Legumes During Development

This study focuses on legume plants grown without rhizobia, enabling the examination of nitrogen metabolism dynamics that reflect the uptake, transport, and assimilation of mineral nitrogen rather than BNF. This experimental framework is especially advantageous for isolating the organisation of mineral nitrogen metabolism within the plant from symbiotic factors, such as nodulation, carbon allocation to nodules, and the export of BNF products. This indicates that nodules did not form on the roots and that nitrogenase did not function. Consequently, variations in nitrate reductase (NR) and nitrite reductase (NiR) activity, as well as the forms of mineral nitrogen, can be interpreted as direct physiological responses to inorganic nitrogen availability. It is important to note that plants received a mixed mineral nitrogen source, predominantly in the form of nitrate. The final hydroponic solution has a NO₃⁻:NH₄⁺ ratio of 10:1. This ratio is crucial to the discussion, as the majority of nitrogen must traverse the assimilatory nitrate reduction pathway, whereas the NH₄⁺ fraction can be assimilated directly, bypassing both NR and NiR.

Research has shown that mineral nitrogen availability can fully support the growth and biomass accumulation of legumes, especially during their early developmental stages. Additionally, while inorganic nitrogen can suppress nodulation and nitrogen fixation, it also promotes root growth and nutrient uptake. This allows legumes to maintain normal physiological function even in the absence of rhizobia.

The uptake of mineral nitrogen by plants is primarily governed by membrane transporters that absorb nitrogen from the solution into the root epidermis and cortex cells, subsequently distributing it throughout the plant’s tissues [51,52]. The primary “gateways” for nitrate (NO₃⁻) are the NRT1/NPF transporters, which typically have a low affinity but serve a broad range of functions, and the NRT2 transporters, which are high-affinity transporters [53,54,55]. Ammonium (NH₄⁺), on the other hand, is taken up by the AMT family of transporters. Once nitrate enters the root cells, it can be: (i) reduced locally within the cells, (ii) temporarily stored (for example, in vacuoles), or (iii) loaded into the xylem for transport to the aboveground parts of the plant [56]. It is in the aboveground organs that further assimilation into usable forms occurs, a process that tends to be more intense. This model of nitrogen uptake—where it is absorbed by the roots and assimilated in the leaves—is particularly likely when the nutrient solution is rich in nitrate and when the leaves have the necessary reducing power to facilitate the further assimilation of nitrite (NO₂⁻) and ammonium (NH₄⁺).

Legumes are often linked with symbiotic nitrogen fixation; however, they are not entirely reliant on rhizobia for their growth. In the absence of these symbiotic bacteria, legumes can take up inorganic nitrogen from the soil or a hydroponic solution, utilising nitrate and ammonium transport systems that function similarly to non-leguminous plants [57]. Higher plants utilise a two-enzyme system for the assimilation of mineral nitrogen. NR located in the cytosol catalyses the conversion of NO₃⁻ to NO₂⁻, while nitrite NiR located in plastids catalyses the reduction of NO₂⁻ to NH₄⁺ [53,56]. The produced NH₄⁺ or transported directly from the hydroponic solution can be rapidly integrated into organic forms via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle, yielding glutamine and glutamate [53,54,55]. These amino acids serve as major nitrogen donors for the biosynthesis of other amino acids, nucleotides, and various nitrogen-containing compounds. Effective assimilation relies on coordination between NR and NiR, as well as on the timely further assimilation of NH₄⁺ [58].

Our results demonstrate a clear organ-specific gradient: NR and NiR activities were highest in leaves, significantly lower in stems, and lowest in roots [59,60]. The distribution of N forms across organs and growth stages was closely associated with the activity of NR and NiR enzymes, indicating coordinated regulation of N assimilation in legume plants grown without rhizobia. In roots, high nitrate concentrations at the V2 stage coincided with elevated NR activity, suggesting active nitrate uptake and initial reduction, whereas consistently low nitrite levels reflected efficient coupling between NR and NiR, preventing nitrite accumulation. As plants progressed to reproductive stages, root NR activity declined despite the presence of nitrate, implying a developmental shift toward nitrate transport to aboveground tissues rather than continued root assimilation. In stems, decreasing NO₃⁻ and NO₂⁻ concentrations from V2 to R3, together with sustained enzyme activities, indicate that this organ functions as a transient site of nitrate processing and redistribution rather than long-term storage. Leaves exhibited the highest NR and NiR activities across all species and growth stages, while nitrate concentrations remained relatively stable, demonstrating rapid nitrate turnover and efficient assimilation. The absence of excessive ammonia accumulation in leaf tissues further supports effective downstream incorporation of reduced nitrogen into organic compounds. This distribution indicates that the primary site of nitrate reduction is the photosynthetic tissues, which is logical, given that leaves have better access to the energy and reducing power required for nitrite reduction in plastids, followed by subsequent NH₄⁺ assimilation. In contrast, roots in this model likely function primarily as nodes for nitrate uptake and transport rather than as major sites for reduction, particularly when nitrogen supply is not limiting, and nitrate can be efficiently transported to the leaves for assimilation [61,62].

The profiles of mineral nitrogen forms (NO₃⁻, NO₂⁻, and NH₃–N) across different organs should be interpreted in conjunction with enzymatic capacity, distinguishing between “quantity” and “flux” [63,64]. Elevated concentrations of NO₃⁻ in tissues do not invariably indicate more intensive assimilation, as NO₃⁻ can be temporarily stored (e.g., in vacuoles) and serve osmoregulatory and reserve functions, especially when it is consistently available in solution. An increase in NO₂⁻ levels within a specific organ or growth stage may indicate a lack of coordination between NR and NiR or limitations in subsequent NH₄⁺ assimilation via GS/GOGAT, leading to the accumulation of this intermediate.

The NH₄⁺ component (approximately 11 mg L⁻¹ in the final solution) adds an additional interpretive layer to the NH₃–N results, as part of the NH₃–N pool may originate not only from the NO₃⁻ → NO₂⁻ → NH₄⁺ pathway but also from direct NH₄⁺ input from the solution [65,66]. Therefore, organs or growth stages with elevated NH₃–N content may reflect not just that nitrate reduction has increased but also temporary NH₄⁺ assimilation loads, variations in transport efficiency, or limitations in carbon skeletons/energy, particularly in non-photosynthetic tissues [67]. This reasoning facilitates a clear distinction in the discussion between: (i) NO₃⁻ accumulation and reduction (linked to NR/NiR), (ii) NH₄⁺ uptake and rapid incorporation into amino acids (GS/GOGAT), and (iii) scenarios where increases in intermediate or final inorganic nitrogen pools result from metabolic coordination rather than nitrogen deficiency or excess.

Ultimately, changes in NR and NiR activities during the V2, R2, and R3 stages, alongside the dynamics of NO₃⁻, NO₂⁻, and NH₃–N, should be analysed in the context of plant development and alterations in nitrogen “sinks,” rather than merely as immediate effects of nitrogen solution concentrations [54,62]. During the vegetative stage, heightened nitrogen demand correlates with the rapid growth of leaves and other vegetative tissues [64]. Conversely, during reproductive stages (flowering and pod initiation), new nitrogen “sinks” emerge, influencing nitrogen distribution and potentially modifying enzymatic activities and inorganic nitrogen pool profiles across organs.

5. Conclusions

Based on the combined analysis of biomass allocation, nitrogen profiles, and NR/NiR activities, sugar pea emerged as the most promising candidate for future combined or intercropping systems with leafy vegetables under hydroponic conditions. This species exhibited high and stable nitrate and nitrite reductase activities in leaves, moderate mineral nitrogen accumulation, and a balanced allocation between shoot and root biomass, indicating efficient nitrogen assimilation without excessive internal nitrogen buffering. Soybean showed similarly strong leaf-based nitrate assimilation but was associated with higher biomass dominance and nitrogen demand, suggesting a potentially stronger competitive role. In contrast, faba bean displayed a more root-oriented nitrogen utilisation strategy and elevated ammonium accumulation, which may limit its suitability for systems prioritising above-ground productivity. Importantly, these interpretations are derived from monoculture hydroponic screening and should be regarded as preliminary indicators rather than direct predictions of intercropping performance.