The Impact of Exogenous Sodium Selenite Treatment on the Nutritional Value and Active Constituents of Pueraria lobata

Abstract

Moderate amounts of Se can promote crop growth, enhance stress resistance, increase yield, and improve nutritional quality. In the present study, kudzu seedlings were used as experimental materials, and their physiological indicators, antioxidant activity, nutritional components, and flavonoid content were measured after being treated with Na₂SeO₃ hydroponics. Transcriptome sequencing analysis was used to reveal the relevant genes involved in regulating the effects of exogenous Se on the content of Se-compounds and flavonoids in kudzu. The results indicated that treatment with 20 mg/L Na₂SeO₃ significantly increased stem and root lengths, dry and fresh weight, lateral root development, and chlorophyll b content. However, at higher concentrations (30–40 mg/L), lateral root abundance and chlorophyll levels decreased. Na₂SeO₃ treatment also augmented the antioxidant capacity and enhanced the content of major nutrients in kudzu seedlings. The total Se content in kudzu escalated with increasing Na₂SeO₃ concentration, with selenomethionine emerging as the primary organic-Se species. After treatment with Na₂SeO₃, the content of puerarin in both aboveground and underground parts decreased, while the content of total flavonoids increased. Daidzin increased in the roots. Differential expression gene analysis revealed that genes such as TRXB2, SYM, MMT1, and METE were involved in Se uptake and transformation in kudzu, while bZIP43 and WRKY47 played a role in flavonoid biosynthesis.

1. Introduction

Selenium (Se) is an essential trace element for animals and humans, playing an important role in the antioxidant system of mammals [1]. Moderate consumption of Se holds significant health benefits for humans. As stipulated by the National Academy of Sciences’ Institute of Medicine in the United States, the recommended daily dietary allowance of Se for adults is 55 µg/day, whereas the tolerable upper intake level is set at 400 µg/day, ensuring adequate intake without adverse effects [2]. People in North America usually consume sufficient Se in their diets and do not face problems related to Se deficiency. However, people living in China, New Zealand, parts of Europe, and Russia occasionally consume insufficient micronutrients, reflecting insufficient Se content in soil and food [3]. Plants grown under acidic and weathered conditions such as tropical soils usually have low Se concentrations. It is estimated that more than 1 billion people worldwide are deficient in Se [4]. Research has found that organic-Se biofortification of crops was safer and more effective than inorganic-Se, and the risk level of inorganic-Se was 40 times higher than that of organic-Se. Furthermore, Se yeast provides a dosage of up to 800 µg/day, which is safe for human consumption [5].

Crops are the primary source of dietary Se intake for humans, and thus, cultivating Se-rich crops is of significant importance for Se supplementation in the human body [6]. In natural environments, Se exists in both inorganic and organic forms, including selenate (SeO₄²⁻), selenite (SeO₃²⁻), selenide (Se²⁻), and elemental Se (Se⁰) in the inorganic state, while organic forms involve selenocysteine (SeCys), methylselenocysteine (MeSeCys), and selenomethionine (SeMet) [7]. Se is primarily utilized by plants in the form of inorganic salts, namely selenite (SeIV) and selenate (SeVI), which are absorbed within plants through sulfate transporters [8]. Selenite enters plants through active transport mediated by phosphate transporters [9]. Plants possess the ability to convert inorganic-Se into organic species, a conversion crucial for enhancing human immune systems, reproductive mechanisms, thyroid function, and the efficiency of intracellular enzymes. Currently, Se-biofortification technology using plants has been widely recognized as an innovative means of cultivating Se-rich agricultural products [10].

While Se is not an essential micronutrient for plant growth, it has been found to promote crop yield, enhance nutritional quality, and strengthen stress resistance in agricultural production at appropriate concentrations [11]. In plants, Se regulates chlorophyll formation and enhances photosynthetic efficiency in leaves by influencing the interaction between two enzymes, 5-aminolevulinic acid dehydratase and porphobilinogen deaminase, which contain -SH groups. This interaction facilitates increased crop yield and optimized quality [12]. Chen et al. [13] observed that a Se concentration of 0.05 mg/L significantly increased chlorophyll content in grape seedlings, while higher concentrations had an inhibitory effect on chlorophyll production. Zhong et al. [14] found that low Se concentrations (≤40 mg/L) promoted photosynthetic pigment accumulation, beneficial to algal growth. Appropriate Se concentrations can also enhance root-reducing power and root vitality, resulting in increased root-shoot ratio, root biomass, and total biomass. The effect of Se on plant growth is concentration-dependent, with low concentrations promoting growth and high concentrations inhibiting it [15]. Cunha et al. [16] explored the effects of Se application on photosynthetic pigments, oxidant metabolism, flavonoid biosynthesis, nodulation, and their relationship with agronomic traits in peanut plants. Se-treated plants exhibited more vigorous growth, increased biomass accumulation in shoots and roots, higher yield, and increased Se concentrations in leaves and seeds.

Furthermore, appropriate Se concentrations can enhance antioxidant enzyme activity in plants and induce the synthesis of non-enzymatic antioxidants such as glutathione, ascorbic acid, proline, flavonoids, and alkaloids, thereby improving the antioxidant capacity of plants. Zhu et al. [17] investigated the effects of foliar spraying with varying concentrations of biosynthesized Se nanoparticles (bio-SeNP) on cabbage and found that all concentrations of bio-SeNP treatment significantly improved plant growth by enhancing the antioxidant defense system (e.g., ascorbic acid glutathione cycle), promoting chlorophyll synthesis, and inhibiting lipid peroxidation products (malondialdehyde, MDA) in shoots. Ahmad et al. [18] demonstrated that exogenous Se application mitigated the negative effects of cadmium stress in mustard plants by regulating osmoprotectants, antioxidant enzymes, and secondary metabolites. Zhang et al. [19] found that Se treatment increased the contents of Se, anthocyanins, and flavonoids in grains. A comprehensive analysis of metabolites and transcriptomes revealed that Se application enhanced the biosynthesis of flavonoids, dihydroquercetin, anthocyanins, and catechins by increasing the expression levels of seven key structural genes (two TaF3H, two TaDFR, one TaF3′5′H, one TaOMT, and one TaANR) involved in flavonoid biosynthesis.

Pueraria lobata (Willd.), commonly known as kudzu, is a leguminous plant with significant pharmacological activities. It possesses important medicinal value and is widely cultivated as a medicinal and edible crop in southern China [20]. Kudzu roots contain a considerable amount of flavonoids, including daidzein, genistein, puerarin, and others. These isoflavones are the primary active ingredients responsible for antioxidant, antidiabetic, and antithrombotic effects [21]. In addition to its rich medicinal components, kudzu also tends to accumulate beneficial mineral elements during cultivation. In Se-rich areas, the Se concentration in kudzu roots can reach up to 70 mg/kg FW, making it an excellent Se-rich food source [22]. Li et al. [23] conducted a comprehensive study to elucidate the molecular mechanisms governing kudzu’s response to Se stimulation. Their findings revealed that regulatory genes play a pivotal role in the biosynthesis of Se-containing compounds and isoflavonoids, in addition to modulating the antioxidant system within kudzu plants. In the present study, wild kudzu was used as the experimental material, and various concentrations of Na₂SeO₃ were applied through hydroponic culture. The biomass, physiological indices, nutritional components, flavonoid content, and different Se speciation were measured. Furthermore, transcriptome sequencing was performed on samples treated with different concentrations of Se, and bioinformatics-based correlation analysis was utilized to evaluate the molecular mechanisms underlying Se absorption, transformation, and the modulation of isoflavonoid content in kudzu. The research data provide theoretical insights into the production and cultivation of Se-enriched kudzu, as well as the enhancement of its nutritional quality and Se biofortification.

2. Materials and Methods

2.1. Plant Materials and Treatment Methods

In this study, water culture treatments with Na₂SeO₃ concentrations of 0, 10, 20, 30, and 40 mg/L were applied to P. lobata beans. The germination trays used had dimensions of 34 cm × 25 cm × 4.5 cm, with a mesh plate aperture of 0.3 cm × 0.3 cm. Each tray was filled with 1 L of 1/2 Hoagland’s nutrient solution to ensure adequate nutrition for the kudzu beans during cultivation. One hundred kudzu beans were uniformly sown in each mesh plate. After 15 days of germination, the corresponding samples were collected and immediately stored at −80 °C for subsequent physiological index measurements. The kudzu seeds employed in the experimental procedures originated from wild kudzu plants harvested in Jianshi County, located within the Enshi Prefecture of China, at geographical coordinates 30°23′ N, 109°72′ E.

2.2. Biomass Measurement

After sample collection, ten seedlings were randomly selected, and the entire weight of each seedling was measured using a precision balance. The average weight was recorded as the fresh weight per plant, expressed in grams. Subsequently, the seedlings were placed in an electric blast drying oven and dried at 65 °C until a constant weight was achieved. The final weight of each seedling was measured using the same precision balance, and the average value was recorded as the dry weight per plant, expressed in grams. The water content was calculated based on the dry and fresh weights, expressed as a percentage (%).

Additionally, after sample collection, ten seedlings were randomly selected, and the lengths of the aboveground and underground parts were measured using a ruler. The average values of these measurements were then calculated, representing the length per plant, expressed in centimeters.

2.3. Determination of the Photosynthetic Pigment Content

Leaves were excised from kudzu seedlings, and 0.1 g of leaf tissue was weighed and placed in a mortar. During the grinding process, 3 mL of 95% ethanol was initially added, followed by the gradual addition of a further 95% ethanol until the tissue attained a white coloration, indicating completion of the extraction procedure. The mixture was then allowed to stand for 3–5 min. After centrifugation at 4000 rpm for 10 min, the supernatant was collected into a volumetric flask and diluted with 95% ethanol to a final volume of 25 mL. The resulting solution was thoroughly mixed and used as the test sample.

The absorbance values of the test sample were measured at wavelengths of 470 nm, 649 nm, and 665 nm, and the following formulas were used for calculations [24]:

C_a = 13.95 × A₆₆₅ − 6.88 × A₆₄₉

(1)

C_b = 24.96 × A₆₄₉ − 7.32 × A₆₆₅

(2)

C_ar = (1000 × A₄₇₀ − 2.05 × C_a − 114.8 × C_b)/245

(3)

C_total = 18.16 × A₆₄₉ + 6.63 × A₆₆₅

(4)

The photosynthetic pigment content (mg/g) was calculated using the following formula:

Photosynthetic pigment content (mg/g) = (C × V × N)/(W × 1000)

(5)

Wherein, C_a denoted the concentration of chlorophyll a (mg/L), C_b represented the concentration of chlorophyll b (mg/L), C_ar signifies the concentration of carotenoids (mg/L), and C_total stood for the total chlorophyll concentration (mg/L). The absorbance values of the test sample at wavelengths 665 nm, 649 nm, and 470 nm are represented by A₆₆₅, A₆₄₉, and A₄₇₀, respectively. C represented the concentration of the respective photosynthetic pigment (mg/L), V denoted the volume of the extracted solution (mL), N signified the dilution factor applied, W represented the fresh weight of the sample (g), and 1000 served as a conversion factor for converting mL to liters.

2.4. Determination of Antioxidant Index

The activity of antioxidant enzymes, including SOD (Kit model AKAO001M), CAT (Kit model AKAO003-2M), and POD (Kit model AKAO005M), were determined using respective detection kits (BOXBIO, Beijing, China). The procedures were conducted according to the manufacturer’s instructions, and the Epoch microplate spectrophotometer (BioTek, Beijing, China) was employed for the quantitative analysis of these antioxidant indices. Specifically, the activity of SOD was quantitatively measured at a wavelength of 560 nm, and the results were expressed in terms of units per gram per minute of fresh weight (U/(g·min) FW), CAT activity was determined at 405 nm and expressed as U/g FW, POD activity was assayed at 470 nm and expressed as U/g FW.

According to the instructions of the reagent kit (Beijing BOXBIO, Kit model AKFA013M), the absorbance values of the reaction sample at 532 nm, 450 nm, and 600 nm were finally converted to the content of MDA and expressed as nmol/g. Similarly, the GSH content was quantitatively assessed using the AKPR008M reagent kit by measuring the absorbance at 412 nm, and the outcomes were subsequently reported in μg/g FW.

2.5. Determination of Nutrient Content

The contents of anthocyanins (Kit model AKPL021M), vitamin C (Kit model AKVI005M), total phenols (Kit model AKPL016M), flavonoids (Kit model AKPL015M), soluble sugars (Kit model AKPL008M), and soluble proteins (Kit model AKPR015) were determined using respective detection kits (BOXBIO, Beijing, China). Following the manufacturer’s instructions, the Epoch microplate spectrophotometer (BioTek, Beijing, China) was utilized to measure the concentrations of these nutritional indices. Specifically, anthocyanins were quantified at 530 nm and 700 nm and expressed as µmol/g FW, ascorbic acid was assayed at 265 nm and expressed as mg/g FW, total phenols were measured at 760 nm and expressed as mg/g FW, flavonoids were analyzed at 510 nm and expressed as mg/g FW, soluble sugars were determined at 620 nm and expressed as mg/g FW, and soluble proteins were quantified at 595 nm and expressed as mg/g FW.

2.6. Determination of the Total Se and Se-Species Content

The total Se content was measured using atomic fluorescence spectrometry (AFS) [25]. Specifically, 0.2 g of the sample was placed in a digestion tube, to which 7 mL of HNO₃ was added and digested in a digestion apparatus. Following digestion, the sample was driven in an acid-driven evaporator at 120–140 °C until the sample volume was reduced to approximately the size of a green bean. The digested sample in the digestion tube was then transferred to a centrifuge tube and diluted with 10% HCl to a final volume of 10 mL for detection. The calculation of total Se content in the kudzu was expressed as

K₁ = (C × V × N)/(W × 1000)

(6)

C represented the mass concentration of total Se (μg/L), V was the total volume of the digested solution for analysis (mL), N was the dilution factor, and W was the dry weight of the sample (g).

To determine the five Se species in the kudzu seedlings, an ultra-high-performance liquid chromatography-mass spectrometry (ICP-MS) system was employed [26]. Five Se standard compounds (selenomethionine (SeMet), MeSeCys, selenocystine (SeCys₂), Se⁴⁺, and Se⁶⁺) were purchased from the National Institute of Metrology, China, for the construction of standard curves. For Se extraction, 0.1 g of kudzu was used, and the sample was hydrolyzed with protease K and protease E. The sample was then sonicated at 37 °C for 1 h and centrifuged at 10,000 rpm for 20 min. The supernatant was filtered through a 0.22 μm filter membrane. The calculation of Se species content in the kudzu was expressed as

K₂ = (C × V × N)/(W × 1000)

(7)

C represented the mass concentration of the Se species (μg/L), V was the total volume of the digested solution for analysis (mL), N was the dilution factor, and W was the dry weight of the sample (g).

2.7. Determination of Flavonoid Content

A precisely weighed 0.5 g sample powder was mixed with 3 mL of 50% methanol solution. The mixture was sonicated at 37 °C for 90 min for extraction. Following extraction, the solution was centrifuged at 10,000 rpm for 20 min, and the supernatant was collected. The supernatant was then filtered through a 0.22 μm organic filter membrane using a syringe, resulting in the test solution.

The Agilent 1260 High-Performance Liquid Chromatography (HPLC, Agilent Technologies, Walldorf, Germany) system equipped with a 1260VWD ultraviolet detector was utilized for the quantitative analysis of isoflavones in kudzu seedlings. The chromatographic separation was achieved on an Agilent Eclipse XDB-C18 column (4.6 mm × 250 mm, 5 μm particle size). A binary gradient elution system was employed, consisting of methanol as mobile phase A and 0.1% formic acid as mobile phase B. Gradient elution was performed as follows: 0–15 min, 28% A; 20–25 min, 28–35% A; 25–30 min, 35–40% A; 30–50 min, 40–75% A; and 50–60 min, 75–28% A. The flow rate was set at 1 mL/min, the wavelength was 250 nm, the column temperature was maintained at 25 °C, and the injection volume was 100 μL.

Standard solutions of puerarin, daidzin, genistin, ononin, daidzein, isoliquiritigenin, and genistein were prepared in 50% methanol with concentration gradients of 0.19–4.75 μg/mL, 0.17–4.25 μg/mL, 0.15–3.75 μg/mL, 0.19–4.75 μg/mL, 0.25–6.25 μg/mL, 0.17–4.25 μg/mL, and 0.14–3.5 μg/mL, respectively. These solutions were then analyzed using HPLC under the previously specified chromatographic conditions. Subsequently, a linear regression analysis was conducted, with the absorption peak area (Y) of each reference substance serving as the dependent variable and the corresponding injection mass (X) as the independent variable. This regression analysis yielded the linear regression equation, correlation coefficient, and linear range for each component. Linear regression equations were derived from these curves. The peak areas obtained from the samples were then substituted into the linear regression equations to determine the respective concentrations of flavonoids in the samples.

The flavonoid content (μg/g) was calculated using the formula:

F_c = (C × V × N)/W

(8)

where C represented the flavonoid concentration (μg/mL), V was the total volume of the test solution (mL), N was the dilution factor, and W was the dry weight of the sample (g).

2.8. Transcriptome Sequencing and Data Analysis

RNA quality was evaluated using the RNA Nano 6000 Assay Kit on the Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA) to determine the total amount and integrity of RNA samples. Following the qualification of RNA libraries, various libraries were pooled based on their effective concentrations and target data volumes prior to submission for sequencing on the Illumina NovaSeq 6000 platform. Paired-end reads of 150 bp were generated. Raw data in Fastq format (raw reads) were first processed through internal Perl scripts. Additionally, the Q20, Q30, and GC content of the clean data were calculated.

Upon obtaining clean reads, Trinity software (v2.6.6) was utilized to assemble the clean reads into reference sequences for subsequent analysis [27]. Feature counts were used to calculate gene alignments, and the FPKM values were subsequently computed based on the length of each gene. Differential expression analysis between two conditions/groups (with two biological replicates per condition) was performed using the DESeq2 R package (1.20.0). The Benjamini–Hochberg method was applied to control the false discovery rate, thereby adjusting the obtained p-values. A threshold of padj < 0.05 and |log2(foldchange)| > 1 was set to identify significant DEGs.

Following the approach outlined by Cheng et al. [28], GOseq (1.10.0) and KOBAS (v2.0.12) software were employed to conduct GO functional enrichment analysis and KEGG pathway enrichment analysis on the differentially expressed genes (DEGs) sets.

Weighted gene co-expression network analysis (WGCNA) was performed on the DEGs using the Metware online cloud platform (https://cloud.metware.cn/#/tools/tool-list, accessed on 20 May 2024) to identify key regulatory genes involved in Se amino acid biosynthesis pathways in response to Na₂SeO₃ treatment. Visualization and statistical analysis were conducted using the R package ggplot2, and a Pearson correlation analysis was performed between the characteristic genes of each identified module and the abundance of Se species. Relevant raw data pertaining to the WGCNA can be accessed and downloaded from the National Genomics Data Center (NGDC) at the following URL: https://ngdc.cncb.ac.cn/omix/submitList, with the specific dataset identifier being OMIX007368 (accessed on 14 September 2024).

2.9. Statistical Analysis

Data analysis was conducted utilizing Excel 2021 (Microsoft, Raymond, WA, USA) and SPSS version 22.0 (IBM, Amonk, NY, USA), with the results expressed as mean values augmented by standard deviations. The graphs were plotted using GraphPad Prism 8 (GraphPad Software Company, San Diego, Ca, USA). The statistical significance of differences among mean values was assessed through the application of Duncan’s multiple range test at a significance level of p < 0.05, and distinct letters were assigned to signify these differences based on the analysis outcomes. Furthermore, a correlation network analysis was executed employing the OmicStudio tools (accessed on 21 June 2024) accessible at https://www.omicstudio.cn/tool, adhering to the methodology outlined by Cheng et al. [29].

3. Results

3.1. Effect of Na₂SeO₃ Treatment on the Biomass of Kudzu

Compared with the control, the shoot length of kudzu seedlings significantly increased after treatment with Na₂SeO₃ (Figure 1a). The shoot length increased by 1.29-fold at 10 mg/L, 2.86-fold at 20 mg/L, 1.24-fold at 30 mg/L, and 0.98-fold at 40 mg/L. Similarly, the root length of kudzu seedlings also increased after treatment with various concentrations of Na₂SeO₃, with increases of 44%, 82%, 57%, and 30% compared to the control.

After Na₂SeO₃ treatment, both the fresh and dry weights of kudzu seedlings were elevated (Figure 1b). Specifically, compared to the control, the fresh weights of seedlings treated with 20 mg/L, 30 mg/L, and 40 mg/L Na₂SeO₃ increased by 97%, 104%, and 63%, respectively. The dry weights increased by 1.28-fold, 1.13-fold, and 72%, respectively. The water content of kudzu seedlings is an important factor affecting plant health. There was no significant difference in water content between kudzu seedlings treated with different concentrations of Na₂SeO₃ and the control (Figure 1c). Compared to the control, the number of lateral roots in kudzu seedlings treated with 20 mg/L Na₂SeO₃ increased by 19%. However, the number of lateral roots in seedlings treated with 30 mg/L Na₂SeO₃ decreased by 12%. There was no significant difference in the effect of other treatments on the number of lateral roots (Figure 1d).

3.2. Impact of Na₂SeO₃ Treatment on the Photosynthetic Pigment Content in Kudzu

Compared with the control, there was no significant change in the total chlorophyll content in the leaves of kudzu seedlings treated with 10 mg/L and 20 mg/L Na₂SeO₃. Significant decreases were observed at concentrations of 30 mg/L and 40 mg/L Na₂SeO₃, with reductions of 33% and 69%, respectively (Figure 2). After treating kudzu seedlings with 10 mg/L and 20 mg/L Na₂SeO₃, there was no significant difference in chlorophyll a content compared to the control group. When the concentration of Na₂SeO₃ was 30 mg/L and 40 mg/L, the chlorophyll a content decreased by 35% and 70%, respectively (Figure 2).

Regarding chlorophyll b, the content in the leaves of kudzu seedlings increased by 17% and 28% compared to the control when treated with 10 mg/L and 20 mg/L Na₂SeO₃, respectively. However, at 30 mg/L and 40 mg/L Na₂SeO₃, the content decreased by 23% and 66%, respectively. Furthermore, compared with the control, the carotenoid content in kudzu seedlings treated with 30 mg/L and 40 mg/L Na₂SeO₃ decreased by 37% and 64%, respectively (Figure 2).

3.3. Effect of Na₂SeO₃ Treatment on Antioxidant Indexes of Kudzu

Compared to the control, the application of 10 mg/L and 30 mg/L of Na₂SeO₃ significantly increased the Vitamin C content in kudzu seedlings by 24% and 53%, respectively. However, there were no significant differences in Vitamin C content between seedlings treated with 20 mg/L and 40 mg/L of Na₂SeO₃ compared to the control (Figure 3a).

Compared to the control, 10 mg/L, 20 mg/L, and 30 mg/L Na₂SeO₃ treatments enhanced the GSH content in kudzu seedlings to varying degrees, with increases of 3%, 2%, and 2%, respectively. Among these, the 40 mg/L treatment showed no significant difference (Figure 3b).

MDA is a biomarker of plant membrane lipid peroxidation under environmental stress. As the concentration of Na₂SeO₃ treatment increased from 20 mg/L to 40 mg/L, the MDA content in kudzu seedlings decreased by 19%, 14%, and 16% (Figure 3c).

Compared with control, the POD activity in kudzu seedlings decreased by 20%, 32%, and 41% after treatment with Na₂SeO₃ at concentrations of 10 mg/L, 30 mg/L, and 40 mg/L, respectively. There was no significant difference in the treatment with 20 mg/L Na₂SeO₃ (Figure 3d). SOD activity in kudzu seedlings significantly increased after treatment with Na₂SeO₃ at concentrations of 20 mg/L, 30 mg/L, and 40 mg/L, by 74%, 122%, and 54%, respectively. There was no significant difference in the treatment with 10 mg/L Na₂SeO₃ (Figure 3e). When the Na₂SeO₃ treatment concentration was 30 mg/L and 40 mg/L, the CAT activity in kudzu seedlings significantly decreased by 49% and 38%, respectively (Figure 3f). Compared with the control group, the APx activity of kudzu seedlings increased by 4.37 times and 3.06 times, respectively, after treatment with 20 mg/L and 40 mg/L Na₂SeO₃. There was no significant difference in other treatments (Figure 3f).

3.4. Effect of Na₂SeO₃ Treatment on the Nutritional Quality of Kudzu

Compared with the control, the soluble sugar content of kudzu seedlings increased by 60%, 49%, and 50% after treatment with Na₂SeO₃ at 10 mg/L, 20 mg/L, and 40 mg/L, respectively (Figure 4a). Compared with the control, treatment with 20 mg/L and 30 mg/L Na₂SeO₃ increased the soluble protein content in kudzu seedlings by 17%, and 16%, respectively. After treatment with 10 mg/L and 40 mg/L Na₂SeO₃, there was no significant difference in soluble protein in the seedlings (Figure 4b). After treatment with 20 mg/L and 30 mg/L Na₂SeO₃, the anthocyanin content in kudzu seedlings increased by 23% and 82%, respectively. However, under treatment with 40 mg/L Na₂SeO₃, there was a reduction of 26% (Figure 4c). In comparison to the control, the application of Na₂SeO₃ treatment resulted in an augmentation of flavonoid content in kudzu seedlings. Specifically, following treatment with 10 mg/L and 30 mg/L Na₂SeO₃, a notable increase of 19% and 15%, respectively, in flavonoid content was observed. However, upon treatment with 20 mg/L and 40 mg/L Na₂SeO₃, a non-significant reduction of 10% and 5%, respectively, in flavonoid content was discerned (Figure 4d).

With regard to total phenolic content, kudzu seedlings exhibited a similar trend of initial increase followed by a decrease as Na₂SeO₃ treatment concentration increased (Figure 4e). Among the different treatments, 10 mg/L, 20 mg/L, and 30 mg/L Na₂SeO₃ led to increases in total phenolic content by 25%, 16%, and 19%, respectively. However, a non-significant decrease of 3% was observed at 40 mg/L Na₂SeO₃ treatment (Figure 4e).

3.5. The Effect of Na₂SeO₃ Treatment on the Content of Total Se and Se-Species

After Na₂SeO₃ treatment, the total Se content in the stems and roots of kudzu seedlings increased with the increase IN exogenous Na₂SeO₃ concentration, but there was no significant difference in the total Se content in the roots between 20 mg/L and 30 mg/L Na₂SeO₃ treatments (Figure 5a). Compared to the control, the Se content in the shoots of kudzu seedlings increased by 30.36-fold, 58.25-fold, 58.15-fold, and 60.35-fold, while the Se content in the roots was 181.20-fold, 272.58-fold, 279.75-fold, and 304.70-fold higher than that of control. Overall, both the shoots and roots of kudzu exhibited enrichment of Se.

Following hydroponic treatment with Na₂SeO₃, SeMet was the primary Se species in the shoots of kudzu (Figure 5b). In the control, no SeCys₂, MeSeCys, Se⁴⁺, SeMet, or Se⁶⁺ was detected in the shoots. Compared to the control, the contents of SeCys₂ and MeSeCys increased with the increasing concentration of Na₂SeO₃, reaching a maximum of 76.2 μg/kg and 83.6 μg/kg at 30 mg/L, respectively. The content of SeMet increased with the increasing concentration of Na₂SeO₃, reaching a maximum of 0.762 mg/kg at 40 mg/L. In comparison to the control group, the maximum concentration of Se⁴⁺ was observed upon treatment with 20 mg/L of Na₂SeO₃, whereas the peak content of Se⁶⁺ was attained following treatment with 30 mg/L of Na₂SeO₃.

In the roots of kudzu after hydroponic treatment with Na₂SeO₃, SeMet was also the primary Se species. Only Se⁴⁺ and SeMet were detected in the control samples (Figure 5c). Compared with the control, the SeCys₂ content in the roots of seedlings increased after treatment with 20 mg/L Na₂SeO₃, reaching a maximum of 0.606 mg/kg. There was no significant difference in SeCys₂ content among the treatments of 20 mg/L, 30 mg/L, and 40 mg/L. The content of MeSeCys and SeMet both increased with the increasing concentration of Na₂SeO₃, reaching the highest at 40 mg/L, which were 1.342 mg/kg and 1.839 mg/kg, respectively. At the root level of seedlings, the highest concentration of Se⁴⁺ was recorded when treated with 20 mg/L of Na₂SeO₃, whereas the maximum content of Se⁶⁺ was observed upon exposure to 30 mg/L of Na₂SeO₃.

3.6. The Effect of Na₂SeO₃ Treatment on the Content of Flavonoids

Puerarin is the primary active component in kudzu. After treatment with Na₂SeO₃ at concentrations of 10–40 mg/L, the puerarin content in the shoots of kudzu seedlings decreased to 253.52 μg/g, 259.47 μg/g, 38.79 μg/g, and 15.85 μg/g. The content of puerarin in the control group was 388.04 μg/g. Compared to the control, the daidzin content in the shoots of kudzu was undetectable after treatment with 10 mg/L and 20 mg/L of Na₂SeO₃. After treatment with 30 mg/L of Na₂SeO₃, the daidzin content increased by 29%, while it decreased by 9% at 40 mg/L. Compared to the control, the genistin content increased by 66% and 36% after treatment with 10 mg/L and 20 mg/L of Na₂SeO₃, but decreased by 38% and 18% after treatment with 30 mg/L and 40 mg/L, respectively. After treatment with 20 mg/L Na₂SeO₃, the content of ononin in shoots of kudzu seedlings increased by 42%, with no significant differences observed in other treatments. Compared to the control, the daidzein content decreased by 24% and 41% after treatment with 10 mg/L and 20 mg/L of Na₂SeO₃, but increased by 14% and 26% after treatment with 30 mg/L and 40 mg/L of Na₂SeO₃. Additionally, isoliquiritigenin was only detected in the sample treated with 10 mg/L of Na₂SeO₃. Compared with the control group, the genistein content decreased by 24% and 38%, respectively, after treatment with 10 mg/L and 20 mg/L Na₂SeO₃ (Figure 6a).

In comparison to the control, the puerarin content in the roots decreased with increasing Na₂SeO₃ concentration, exhibiting a reduction of 87%, 98%, 97%, and 99%, respectively. The daidzin content increased in all Na₂SeO₃-treated samples, exhibiting an increase of 5.41-fold, 4.3-fold, 2.22-fold, and 3.38-fold compared to the control. The genistin content initially increased and then decreased, with an 11.01% increase at 10 mg/L but a decrease of 41%, 37%, and 40% at 20 mg/L, 30 mg/L, and 40 mg/L, respectively. After Na₂SeO₃ treatment, the ononin content decreased by 28%, 52%, 38%, and 25%. Compared with the control, the content of daidzein increased by 18%, 21%, and 16% after treatment with Na₂SeO₃ at 20 mg/L, 30 mg/L, and 40 mg/L, respectively. The isoliquiritigenin content decreased by 47%, 44%, 57%, and 57% compared to the control. The genistein content decreased by 29%, 46%, 38%, and 35% compared to the control (Figure 6b).

3.7. The Effect of Na₂SeO₃ Treatment on the Transcriptome Data of Kudzu

The Illumina HiSeq sequencing was performed on the seedlings of kudzu treated with Na₂SeO₃. For each sample, triplicate sequencing was conducted, and the Pearson correlation coefficients between replicates were mostly above 0.8, with higher correlation coefficients observed between replicates than between non-replicates (Figure S1a). Compared to the control, the number of upregulated genes after treatment with 10–40 mg/mL of Na₂SeO₃ were 10, 11, 168, and 37, while the number of downregulated genes were 12, 17, 82, and 52, respectively (Figure S1b).

To identify gene co-expression modules related to Se metabolism in kudzu seedlings, WGCNA was performed on all expressed genes. After filtering out lowly expressed genes, 16,551 genes were retained for network construction. When the correlation coefficient R² was set to 0.9 and the soft threshold was 15, 27 modules were generated after merging similar clusters (Figure S2). The gray module, containing genes with a chaotic expression pattern, was excluded from further analysis (Figure 7).

Furthermore, association analysis was performed between Se species content as traits and DEGs (Figure 8a). The trait-module correlation heatmap revealed that the genes in the dark turquoise module were significantly positively correlated with methylselenocysteine and hexavalent Se. The genes in the light green module were positively correlated with various Se species in kudzu, while the genes in the magenta module were negatively correlated with all six Se species (Figure 8b). Subsequently, the dark turquoise, light green, and magenta modules were selected for further analysis.

GO and KEGG enrichment analyses were performed on these three modules. The genes were primarily involved in three categories of functions: Biological Process, Cellular Component, and Molecular Function. GO terms in the dark turquoise module were mainly enriched in biological processes related to carotenoid biosynthesis and metabolism, as well as in cellular components such as chloroplasts, chloroplast outer membranes, and plastid outer membranes (Figure S3a). KEGG enrichment analysis revealed involvement in various secondary metabolic pathways (Figure S4a). The GO terms in the light green module were primarily enriched in molecular functions related to poly(A) RNA binding, polypurine binding, and exogenous transmembrane transporter ATPase activity (Figure S3b). KEGG analysis revealed enrichment in aflatoxin biosynthesis pathways (Figure S4b). The GO terms in the magenta module were primarily enriched in molecular functions such as metal ion transmembrane transporter activity, protein cysteine S-palmitoyltransferase activity, and protein cysteine S-acyltransferase activity (Figure S3c). KEGG enrichment was observed in various O-glycan biosynthesis pathways (Figure S4c).

3.8. Screening of Hot Genes Related to Se-Species Metabolism

The expression heatmap results for genes in the dark turquoise module revealed significant upregulation of gene expression following treatment with 30 mg/L and 40 mg/L of Na₂SeO₃ (Figure S5a). In the light green module, expression levels only increased under 40 mg/L Na₂SeO₃ treatment, while they were downregulated under other treatments (Figure S4b). In the magenta module, expression levels increased only in the control group and were downregulated under other treatments (Figure S5c).

Based on the relevant literature, key genes related to Se metabolism were identified from the dark turquoise, light green, and magenta modules, including thioredoxin reductase (TRXB2), methionine-tRNA ligase (SYM, SYMM), methionine S-methyltransferase (MMT1), and homocysteine methyltransferase (METE). Correlation analysis was performed between these three modules and total Se content as well as Se species content, and key genes with high correlation were screened out. Functional annotations were provided for these key genes using data from the NCBI database (Table S1).

A total of 60 hotspot genes were screened from the three modules, and correlation analysis was performed between these hotspot genes and total Se content as well as Se species content (Table S2). A correlation heatmap was generated. The results showed that the DEGs GLYMA_08G217400 (NIP61) had the strongest positive correlation with Se content, followed by GLYMA_20G090000 (PHYA2). Conversely, the gene GLYMA_18G277100 (BZP43) exhibited the strongest negative correlation with Se content (Figure 9a). Subsequently, a correlation network diagram was constructed for total Se content and Se species content. All Se species showed a positive correlation with total Se. In terms of radiation targets, the gene GLYMA_08G217400 (NIP61) had the highest number of related targets, totaling 14, including total Se content, organic Se content, and genes related to Se metabolism. GLYMA_20G090000 (PHYA2) had 11 related targets, including total Se, organic Se, and SeCys. GLYMA_08G165700 (NFYC2) had 9 related targets (Figure 9b).

3.9. Correlation Analysis between Flavonoid Content and DEGs

After Na₂SeO₃ treatment, WGCNA analysis was performed on the contents of genistin, genistein, puerarin, daidzein, daidzin, ononin, and isoliquiritigenin in samples from different treatment groups, and a total of 27 modules were obtained (Figure S6a). Among them, the turquoise module contained the largest number of genes, totaling 2143, while the dark orange module had the fewest genes, with only 68 genes (Figure S6b). The trait-module correlation heatmap revealed a positive correlation between the black module and these seven flavonoids, while the magenta module showed a high correlation across all substances. The black and magenta modules clustered 1059 and 1189 genes, respectively. Therefore, the black and magenta modules were selected as target modules for further analysis (Figure S6c).

The correlation heatmap analysis between core genes and flavonoid content showed that GLYMA_08G297400 (WRKY14) had the strongest positive correlation with puerarin content, followed by GLYMA_13G342700 (4CL6) (Figure 10a). In contrast, GLYMA_10G284700 (ARGJ) exhibited the strongest negative correlation with daidzein content. Figure 10b depicts the correlation network of core genes and flavonoid content (connectivity absolute value > 0.7). The results indicated that the gene GLYMA_15G108700 (BH051) had the highest number of related targets, totaling 15. GLYMA_15G127200 (NPR1) had 10 related targets, and GLYMA_08G042600 (ATG16) had 9 related targets.

4. Discussion

4.1. The Effect of Se on the Growth of Kudzu

The application of appropriate levels of Se in plants is conducive to their growth and development, resulting in modifications to both the aboveground and belowground components. Compared to the control, the addition of low concentrations of Na₂SeO₃ led to larger kudzu seedlings and longer thicker roots. This suggests that low concentrations of Na₂SeO₃ stimulate the growth of kudzu seedlings, whereas higher concentrations inhibit it, manifesting as an initial increase and subsequent decrease in root hair number, as well as a thinning of the main root compared to the control. However, the dry and fresh weights of the seedlings increased. Optimal concentrations of Se promote the growth of broccoli plants, balance hormonal levels and nutrient distribution, reduce water content, and stimulate root growth, fresh weight, and dry weight [30]. When mung bean plants were treated with Na₂SeO₃ at concentrations below 50 mg/kg, a significant increase in stem elongation and root marginal expansion was observed [29]. Abdoun et al. [31] found that foliar application of Se at concentrations of 0, 12.5, and 25 mg/L resulted in an increase in moringa plant height, leaf area, and stem dry and fresh weights as Se levels increased. Overall, the application of 20 mg/L Na₂SeO₃ yielded the highest stem length, the greatest number of lateral roots, and the highest dry weights in the plants, indicating that this concentration is the optimal level for the hydroponic cultivation of kudzu.

Photosynthesis, a vital process for plant growth, involves the utilization of photosynthetic pigments to convert CO₂ and water into organic compounds under visible light, with the transformation of light energy into chemical energy. Araujo et al. [32] observed that Se application enhanced the chlorophyll and carotenoid concentrations in sugarcane plants, particularly when treated with 4 mg/L Na₂SeO₃, resulting in higher total chlorophyll content during the peak flowering stage. Different plant species exhibit varying tolerance to Se concentrations. For instance, in coffee plants, the addition of Se up to 40 mg/L increased the concentration of photosynthetic pigments in the leaves [33]. After treatment with 10 mg/L and 20 mg/L Na₂SeO₃, the chlorophyll a content had no significant difference with the control, and the chlorophyll b content increased by 17% and 28%, respectively. However, at higher concentrations, the contents of chlorophyll a, chlorophyll b, and carotenoids decreased, indicating that kudzu has a lower tolerance threshold for selenite than 40 mg/L. Therefore, in this study, low concentrations of Na₂SeO₃ significantly improved the growth performance of kudzu seedlings, shortened the seedling stage, and increased the yield. Conversely, excessive concentrations of Na₂SeO₃ inhibited plant growth and reduced photosynthetic pigment content.

In wheat, Se-induced oxidative stress occurs at high concentrations, leading to tissue damage and death. Under such conditions, the levels of hydrogen peroxide and antioxidant enzyme activities increase significantly, enabling the plants to establish an antioxidant defense system to adapt to selenite stress [34]. Ren et al. [35] observed an increase in Vc content and enhanced antioxidant capacity in Fuji apples treated with 100–150 mg/L Na₂SeO₃ spray. Similarly, in our experiments, Na₂SeO₃ treatment increased Vc content in kudzu seedlings, contributing to an enhancement in their antioxidant capabilities.

Furthermore, low concentrations of Na₂SeO₃ have been found to increase glutathione (GSH) content and decrease MDA levels in some crops [36]. For instance, in Chlorella vulgaris, 10 µM of Na₂SeO₃ treatment resulted in elevated GSH levels and reduced MDA levels [37]. In rice, treatment with 0.5–20 mg/kg Na₂SeO₃ significantly increased GSH content, glutathione peroxidase (GPX) activity, and peroxidase (POD) activity [36]. In kudzu, various concentrations of Na₂SeO₃ treatment led to varying degrees of increase in GSH content, while MDA content was consistently lower than the control. These findings indicate that selenite treatment positively affects antioxidant enzyme activities in different crops. For example, in mustard plants treated with different concentrations of Na₂SeO₃, antioxidant enzyme activities exhibited alternating increases. Specifically, at a Na₂SeO₃ concentration of 10 mg/L, SOD activity and GSH content were the highest, while at 20 mg/L, POD and CAT activities peaked [15]. In kudzu, Na₂SeO₃ treatment enhanced SOD and APx enzyme activities, but POD and CAT enzyme activities decreased. This suggests that antioxidant compounds such as GSH and Vc, as well as antioxidant enzymes SOD and APx, play crucial roles in scavenging reactive oxygen species under Na₂SeO₃-induced oxidative stress.

4.2. The Effect of Se on the Nutritional Quality of Kudzu

In addition to enhancing crop yield and antioxidant properties, low concentrations of Se also affect the nutritional quality of crops. When winter jujube was sprayed with 50 mg/L of Na₂SeO₃, compared to the untreated control, the Vc content increased by 20.94%, soluble sugar content by 29.48%, total flavonoid content by 43.48%, and the sugar-acid ratio by 41.81%. However, as the concentration of Na₂SeO₃ increased, the yield and quality of winter jujube began to decline [38]. Treatment with 0.25–0.50 mg/L of Na₂SeO₃ significantly increased the content of phenolic compounds, flavonoids, and anthocyanins in wheat [39]. In broccoli, Se yeast and selenite significantly increased the content of total phenolic acids and glucosinolates in the leaves, but Se yeast reduced the total flavonoid content [40]. The impact of Na₂SeO₃ treatment on the nutritional quality of kudzu seedlings is complex. Under hydroponic conditions, various concentrations of Na₂SeO₃ resulted in an upward trend in the content of soluble sugars, soluble proteins, flavonoids, and total phenols in the seedlings. However, anthocyanin content only increased significantly at a treatment concentration of 30 mg/L.

In addition to the aforementioned nutritional components, the content of trace elements, especially Se, is a crucial aspect of the nutritional quality of crops. Applying Na₂SeO₃ during critical growth stages of rice significantly increases the total Se and organic Se content. Treatment with 10 mg/L Na₂SeO₃ results in the highest organic Se content (0.03 mg/kg) in brown rice, followed by the embryo, rice bran, and finally, the hull. Furthermore, 75–85% of the Se in brown rice and embryos is in an organic form [41]. Following Na₂SeO₃ treatment, five Se species were identified in dandelion seedlings, including Se⁴⁺, Se⁶⁺, SeCys₂, SeMet, and MeSeCys, with SeMet being the primary organic Se species in the aboveground parts [28]. Treatment of broccoli with yeast Se and Na₂SeO₃ significantly increased the total Se content in the heads, with SeMet and MeSeCys being the primary Se species [40]. The bioavailability of different Se species differs: SeMet, SeCys, selenate, and selenite exhibit gradually decreasing bioavailability [42]. Appropriate Na₂SeO₃ treatment enhances the total Se and organic Se content in kudzu seedlings, with organic Se primarily consisting of SeMet, SeCys₂, and MeSeCys.

Puerarin, the primary active component in kudzu, possesses significant medicinal and health benefits [43]. Se treatment typically promotes the accumulation of certain bioactive substances in plants, though high Se concentrations can exert inhibitory effects. For instance, nano-Se and organic-Se treatments have been shown to enhance flavonoid content in Ginkgo biloba leaves [44]. Low concentrations of Na₂SeO₃ treatment in soybeans significantly increased total phenols, isoflavones, and amino acid content, while higher Se concentrations exhibited inhibitory effects [45]. Similarly, 4 μM and 8 μM Na₂SeO₃ treatments significantly increased anthocyanin content in purple-leaf lettuce, but anthocyanin content was significantly reduced at a higher Se concentration of 16 μM [46]. After Na₂SeO₃ treatment, the content of total flavonoids in kudzu seedlings increased, but the content of puerarin decreased. At the concentration of 30 mg/L Na₂SeO₃, the content of daidzin was only higher than that of the control group, and the content of daidzin in the underground part was always higher than that of the control group. This preliminary observation showed that the experimental materials were in the seedling stage of kudzu and accumulated relatively less puerarin. However, flavonoids upstream of the biosynthetic pathway respond to the stimulation of Se, leading to the accumulation of daidzin.

4.3. Absorption and Transformation of Se in Kudzu

Se and sulfur belong to the same group of elements, exhibiting similar chemical properties. Se is often transported in plants through sulfur metabolic pathways. In plants, cystathionine γ-synthase (CGS), methionine methyltransferase (MMT), and METE are key enzymes involved in the synthesis of methionine through the aspartate (Asp) family pathway and the S-methylmethionine (SMM) cycle [47]. The SeMet cycle is a significant pathway for Se metabolism, and enzymes such as S-adenosylmethionine synthase (MAT), SAM-dependent methyltransferase (MTase), S-adenosylhomocysteine hydrolase (SAHH), and methionine synthase (MTR) that participate in this cycle have a higher affinity for Se metabolites than sulfur metabolites [48]. In the paper mulberry tree (Broussonetia papyrifera), genes differentially expressed in the Na₂SeO₃ treatment group were significantly enriched in 24 metabolic pathways, with sulfur assimilation genes being upregulated. Additionally, Se-binding proteins and transporter genes, such as SBP1, PCS, GSTs, ABCs, and GPX, were significantly induced after selenate treatment [49]. In alfalfa, the ABC transporter G36, glutathione S-transferase, and cysteine-tRNA ligase activity were affected by Na₂SeO₃ treatment [50]. SYM plays a crucial role in Se accumulation and tolerance in soybeans [51]. In kudzu treated with Na₂SeO₃, core genes TRXB2, SYM, MMT1, and METE, identified from the dark turquoise, light green, and magenta modules, were involved in Se absorption and transformation. NIP6-1, which is associated with boron absorption, transports it to aboveground plant parts, promoting its effective absorption and utilization [52]. In rice, Se alleviates arsenic toxicity in seedlings by regulating the expression of genes such as NIP1;1 and NIP2;1 [53]. The phytochrome PHYA homolog regulates the core flowering inhibitor E1 at transcriptional and post-transcriptional levels [54]. NFYC2, a component of the NF-Y complex, participates in various cellular processes, including DNA damage repair, cell cycle regulation, and abscisic acid signaling, playing a crucial role in coordinating stress responses [55]. In this study, the enhanced expression of NIP61 in kudzu seedlings treated with Se may be related to the abiotic stress caused by Na₂SeO₃, participating in the adaptation to Se stress during development. The responses of PHYA2 and NFYC2 in kudzu seedlings to Se treatment may be associated with nucleic acid damage repair and normal photosynthesis in chloroplasts.

4.4. The Effect of Se on the Content of Flavonoids in Kudzu

Puerarin, daidzin, genistin, ononin, daidzein, and genistein belong to the isoflavone class, while isoliquiritigenin belongs to the chalcone class. Isoflavones are synthesized through a series of reactions catalyzed by enzymes such as L-phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone reductase (CHR), 2-hydroxyisoflavanone synthase (IFS), 2-hydroxyisoflavanone dehydratase (HID), and UDP-glucosyltransferase (UGT). At the transcriptional level, the biosynthesis of isoflavones is mainly regulated by structural genes (SGs) and transcription factors (TFs). Six PtR2R3-MYB genes and seven structural genes (PtHID2, PtHID9, PtIFS3, PtUGT069, PtUGT188, PtUGT286, and PtUGT297) directly or indirectly regulate the biosynthesis of puerarin [56]. After treating alfalfa with Na₂SeO₃, it was found that DEGs were mainly enriched in the MAPK signaling pathway, phenylpropanoid biosynthesis, isoflavonoid biosynthesis, cutin, suberin, and wax biosynthesis, and glycerolipid metabolism [57]. Zhang et al. [19] found that Se treatment increased the total content of Se, anthocyanins, and flavonoids in grains. Through comprehensive analysis of metabolites and transcriptomes, they discovered that Se application enhanced the biosynthesis of flavonoids, dihydroquercetin, anthocyanins, and catechins by increasing the expression levels of seven key structural genes in flavonoid biosynthesis (two TaF3H, two TaDFR, one TaF3′5′H, one TaOMT, and one TaANR). The bZIP43 transcription factor participates in anthocyanin accumulation by regulating the expression of anthocyanin biosynthesis structural genes [58]. In A. thaliana, the WRKY47 played an important role in maintaining Se homeostasis and tolerance. The expression of WRKY47 was induced by Se stress [59]. Additionally, specific WRKY proteins played a crucial role in regulating flavonoid metabolism to enhance plant stress adaptation [60]. Therefore, when kudzu seedlings were treated with Na₂SeO₃, the expression of bZIP43 and WRKY47 was strongly associated with flavonoid content, suggesting their involvement in stress responses caused by abiotic stress and also participating in the synthesis of kudzu isoflavones.

Basic helix-loop-helix (bHLH) motifs constitute one of the largest TF families and play a significant role in regulating various secondary metabolites. The interaction between bHLH3 and MYB4 ensures the accumulation of flavonoids required for mulberry fruit development and is also associated with anthocyanin synthesis [61]. In higher plants, 4CL is a key enzyme in flavonoid metabolism, catalyzing the conversion of various hydroxycinnamic acids into their corresponding CoA esters, thus directly participating in phenylpropanoid metabolism and subsequently synthesizing flavonoids and anthocyanins [62]. Salicylic acid (SA) is a defense hormone that regulates NPR1, the primary transcriptional regulator of immune-related genes [63]. Furthermore, the synergistic action of SA-mediated genes may be related to the expression of flavonoid biosynthesis pathway genes (FLS, CHS, CHI, F3H, and ANS), leading to increased phenolic compound content in SA-pretreated plants [64]. In this study, Se treatment enhanced the accumulation of flavonoids and anthocyanins in kudzu, potentially due to the elevated SA levels and NPR1 expression induced by stress. Consequently, NPR1 expression regulates the downstream synthesis of flavonoids and anthocyanins.

5. Conclusions

Low concentrations of Se, specifically 20 mg/L Na₂SeO₃ treatment, were advantageous in enhancing the growth attributes of kudzu seedlings. This manifested in the augmentation of stem length, root length, and lateral root number, alongside a marked increase in both dry and fresh weights. Notably, treatments with 20 mg/L and 30 mg/L Na₂SeO₃ had a significant impact on chlorophyll b content, whereas higher concentrations of Na₂SeO₃ exhibited an inhibitory effect on photosynthetic pigment content. Additionally, Na₂SeO₃ treatment upregulated the activities of SOD, APx, and GSH enzymes, while concomitantly reducing the levels of MDA. Compared to the control, Na₂SeO₃ treatment increased the content of major nutrients in kudzu seedlings, with the total Se content increasing in a concentration-dependent manner. SeMet was identified as the primary organic-Se species in kudzu. Treatment with Na₂SeO₃ led to a decrease in puerarin content in both the shoots and roots of the seedlings, but an increase in daidzin and total flavonoid content. After Na₂SeO₃ treatment, TRXB2, SYM, MMT1, and METE genes were involved in the absorption and transformation of Se in kudzu. In addition, NIP61 played a role in the tolerance and adaptation to Se stress during seedling development. bZIP43 and WRKY47 were involved in the synthesis of flavonoids in kudzu seedlings.