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Article

Transcriptome and Metabolome Revealed Impacts of Zn Fertilizer Application on Flavonoid Biosynthesis in Foxtail Millet

by
Ke Ma
1,2,
Xiangyu Li
1,
Xiangyang Chen
3,
Chu Wang
1,
Zecheng Zhang
1,
Xiangyang Yuan
4,
Fu Chen
1,2 and
Xinya Wen
1,2,*
1
College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
2
Key Laboratory of Farming System, Ministry of Agriculture and Rural Affairs of China, Beijing 100193, China
3
Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs of the People’s Republic of China, Beijing 100176, China
4
College of Agronomy, Shanxi Agricultural University, Taigu, Jinzhong 030801, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(8), 1767; https://doi.org/10.3390/agronomy15081767
Submission received: 13 June 2025 / Revised: 9 July 2025 / Accepted: 21 July 2025 / Published: 23 July 2025
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

To explore the effects of various zinc (Zn) fertilizer application methods and concentrations on foxtail millet quality and flavonoid biosynthesis, we used Zhangzagu 13 as the experimental material. The transcriptome and metabolome were used to examine variations in flavonoid biosynthesis and metabolism in foxtail millet under different Zn application methods. The results showed that different Zn application methods significantly increased the total polyphenol, carotenoid, total flavonoid, and Zn contents in the grains of foxtail millet. Under the basal soil application (S3) and foliar spray (F2) treatments, the total flavonoid content significantly increased by 45.87% and 64.40%, respectively, compared with that of CK. Basal soil Zn fertilization increased the flavonoid content of foxtail millet by up-regulating genes related to flavonoid metabolism and biosynthesis, including flavanone-3-hydroxylase, chalcone isomerase, and leucoanthocyanidin reductase. Foliar Zn application enhanced flavonoid content by up-regulating genes involved in flavonoid metabolic and biosynthetic processes and chalcone isomerase. In conclusion, using Zn fertilizer can improve the synthesis and metabolism of foxtail millet flavonoids, effectively increase the content of functional substances in grains, and realize the biofortification of foxtail millet grains.

1. Introduction

Mountains cover approximately 70% of China’s land area. Compared with flat and open plains that are suitable for large-scale mechanized farming, mountainous areas are at a significant disadvantage in developing bulk grain and oil crops, resulting in products that lack market competitiveness [1,2]. Hence, the evolution of agriculture in mountainous areas should adhere to the principle of adapting to local conditions, leveraging their advantages, and adjusting the industrial structure [3]. Because of their strong drought resistance and stress tolerance, most coarse grains grow in remote mountainous areas, high-altitude regions, and economically underdeveloped areas [4]. The perennial cultivation area of coarse grains in China ranges from 8 to 10 million hectares, with a total annual output of approximately 16 to 20 million tons, accounting for 6–8% of the total grain production. With the continuous improvement in China’s material living standards, coarse cereals, which are rich in nutritional value and health care functions, account for an increasing proportion of people’s food structures.
Foxtail millet (Setaria italica L.) is a vital coarse-grain crop characterized by its rich historical and cultural significance, high nutritional value, drought and barren soil tolerance, environmental friendliness, and dual-purpose use as both food and fodder [5,6]. Foxtail millet is mainly distributed in 10 provinces and cities, including Shanxi, Hebei, Inner Mongolia, Shaanxi, Liaoning, Henan, Jilin, Shandong, Heilongjiang, and Gansu, covering 97% of the total national area. Among these locations, 60% of foxtail millet is distributed in Inner Mongolia, Shanxi, and Hebei. Recently, the nutritional components and antioxidant capacity of foxtail millet have attracted widespread attention. Foxtail millet is rich in bioactive substances that play an important role in reducing the rate of fat absorption, slowly releasing sugars, and decreasing the risk of diabetes, hypertension, and heart disease [7,8]. Flavonoids, including flavones, flavanones, flavonols, isoflavones, and anthocyanins, are an important class of secondary metabolites in foxtail millet. They have various biological functions, including antioxidant and anti-inflammatory functions [9,10].
The application of zinc fertilizer can influence plant growth, development, and secondary metabolic processes [11]. For basal soil application, exogenous zinc is primarily taken in by plant roots through Zrt- and Irt-like protein (ZIP) transporters. Following root uptake, Zn2+ is loaded into the xylem for transport to shoots, a process mediated by heavy metal ATPase (HMA) family proteins [12]. Within cells, Zn2+ is compartmentalized into vacuoles via metal tolerance protein (MTP) transporters and stored complexed with phytochelatins (PCs) [13]. Zinc is also integrated into metallothioneins and various zinc-dependent enzymes, leading to elevated levels of PCs, metallothioneins, and functional proteins [14]. The biofortification of zinc in edible parts (e.g., grains) is achieved by enhancing the expression of grain-localized HMA/MTP transporters and reducing the phytic acid content [15]. In foliar spray applications, zinc fertilizers enter leaf mesophyll cells either by diffusion through the cuticle or by stomatal uptake [16]. ZIP transporters facilitate intracellular transport within leaf cells [17]. Zinc ions are subsequently compartmentalized into vacuoles by MTPs (where they can be chelated by PCs). A portion of the absorbed zinc is remobilized to developing grains via the phloem, enabling a rapid increase in grain zinc concentration [18]. Additionally, previous studies have shown that in cowpea grains, zinc fertilization may increase the content of zinc and storage proteins while decreasing that of phytic acid [19]. The tocopherol, zinc, and crude protein contents of maize were significantly increased by zinc treatment [20]. Applying zinc to rapeseed increased the levels of total oil, oleic acid, and linoleic acid while decreasing the levels of stearic acid [21]. Additionally, Zn-treated fruits had fewer flavonoids than CK [22]. In contrast, Zn treatment increased the flavonoid content of Artemisia annua L. [23]. However, the effects of Zn treatment on the content of nutrients and the physiological metabolism of flavonoids in foxtail millet remain unclear. Furthermore, numerous studies have shown that different Zn fertilizer application methods (leaf spraying, soil-based application, and seed treatment) have different effects on the yield and grain quality of crops [24].
Based on the preceding mechanisms, we hypothesized that Zn fertilizer application would influence the nutritional composition of foxtail millet grains and that different Zn application methods (soil vs. foliar application) would exhibit differential involvement in the biosynthesis and metabolism of flavonoids. This study aimed to explore the following: (1) the effects of different Zn fertilizer application methods and Zn concentrations on foxtail millet quality; (2) the effects of different Zn fertilizer application methods on the flavonoid biosynthesis pathway of foxtail millet; and (3) the significance of Zn fertilizer application in foxtail millet bioaugmentation.

2. Materials and Methods

2.1. Experimental Station Description

This experiment was conducted at the organic dry farming experimental station of China Agricultural University (39°52′19″ N, 113°29′32″ E). The altitude was 1009.5 m, the average annual temperature was 6.4 °C, the average precipitation was 391.5 mm, and the average frost-free period was 125 days. The soil type in the experimental field was sandy loam, and the crop planted the previous year was foxtail millet. The physical and chemical characteristics of the soil in the experimental field were determined before sowing (Table S1).

2.2. Experimental Materials

“Zhangzagu 13” was used in this experiment, which was bred in Zhangjiakou City, Hebei Province. The benefits of Zhangzagu 13 include its high yield, nutrient-dense diet, and environmental tolerance. The type of zinc fertilizer used in soil-based application and foliar application was ZnSO4·7H2O.

2.3. Experimental Design

The field experiment was carried out in 2023. We used a two-factor random block design, and the two factors were the following: A zinc application method (basal soil application and foliar application); B zinc fertilizer ZnSO4·7H2O dosage. A total of eight treatment combinations were used, and the specific treatment is shown in Table S2. The base application treatment involves adding zinc fertilizer into the soil before sowing, and the spray treatment is conducted during the early stages of foxtail millet grain filling in the evening when there is no wind. There were three replicates for each treatment, for a total of 24 plots.
Before sowing, base fertilizer was applied in combination with the preparation of the field. The fertilizer used was a high-nitrogen and high-potassium compound fertilizer, in which the N content was 25%, the P2O5 content was 10%, the K2O content was 16%, and the application amount was 600 kg/hm2. The plants were sown on 10 May 2023 and harvested on 28 September 2023. Bed seeding was performed using drip irrigation and a film-mulching machine. Dripped irrigation was used as the water management method, and other conventional local management measures were adopted.

2.4. Sample Collection

Grains were sampled at maturity, and each replicate was taken by combining the foxtail millet grains of five spikes from the same field plot. A total of 24 samples were harvested and threshed, using spikes from three different field plots with three biological replicates. Based on the observation that the S3 and F2 treatments exhibited the greatest increase in flavonoid content and represent distinct zinc uptake dynamics, S3 (basal soil application) and F2 (foliar spray) were selected for transcriptomic and metabolomic analyses. Additionally, the S3 and F2 samples were promptly frozen in liquid nitrogen and stored at −80 °C for RNA extraction, RNA-Seq, and metabolome analysis in the subsequent studies.

2.5. Methods

2.5.1. Measurement of Grain Quality

Referring to Chen [25], who used near-infrared nondestructive testing technology, fat, carbohydrate, protein, and total amino acid contents were determined using a near-infrared spectroscopy analysis model (NIRS TM DS2500, FOSS, Hillerød, Denmark).
The amylose content was quantified following the method described by Liu et al. [26], with minor modifications. Briefly, 20 mg of foxtail millet flour was accurately weighed into a 20 mL test tube. Then, 0.2 mL of 95% ethanol was added to this to wet the sample, followed by 1.8 mL of 1 mol·L−1 NaOH added along the tube wall. The mixture was vortexed and then incubated in a boiling water bath for 10 min. After cooling to room temperature, the volume was adjusted to 20 mL with distilled water and thoroughly mixed. An aliquot of 0.5 mL of this solution was transferred to a 10 mL centrifuge tube containing 5 mL of distilled water. Subsequently, 0.1 mL of 1 mol·L−1 acetic acid was added, followed by 0.2 mL of iodine reagent (0.2% I2 and 2% KI) and 4.2 mL of distilled water. The solution was mixed, and after 10 min, the absorbance at 720 nm was measured using a spectrophotometer (Sunny Heng ping Instrument, LLC. Shanghai, China). Amylose content was calculated based on a standard calibration curve, with a distilled water blank used for zeroing the instrument.
The yellow pigment, total polyphenol, and total flavonoid contents were determined according to Ma et al. [27].
Zinc content was measured using flame atomic absorption spectrometry (iCE 3000, Thermo Fisher Scientific, Waltham, MA, USA). A 0.500 g sample was accurately weighted, and 5 mL of HNO3 and 1 mL of H2O2 were added to the microwave digestion tube. Only HNO3 and H2O2 were added into the blank tube. A microwave digestion tube was placed in a microwave digestion instrument for sample digestion. After cooling, distilled water was added to a 25 mL volumetric bottle containing the digesting and washing liquids, and the mixture was mixed for measurement. The liquid to be measured was introduced into a flame atomizer, the absorbance value was measured after atomization, and the zinc content was calculated using a standard curve.

2.5.2. Metabolome Analysis

The foxtail millet grain sample was freeze-dried using a freeze dryer (SCIENTZ-100F) under vacuum conditions (10–100 Pa), with the condenser temperature reaching a minimum of −55 °C. Freeze-dried foxtail millet samples were ground into a powder for 1.5 min at 30 Hz. Then, 50 mg of samples was accurately weighed and added to 1200 μL of 70% methanol water internal standard extraction solution pre-cooled at −20 °C. The samples were vortexed six times for 30 s each and centrifuged at 12,000 rpm for 3 min, and the supernatant was filtered and stored for ultra-performance liquid chromatography–tandem mass spectrometry analysis.
The chromatographic and mass spectrometry conditions are listed in Table S3. The substance was evaluated using secondary spectrum data and quantified using multiple reaction monitoring, using the multi-reaction monitoring mode of triple quaternary bar mass spectrometry, based on the Wuhan Maiwei Metabolism Company Metware database.

2.5.3. Transcriptome Analysis

RNA extraction and transcriptome sequencing library preparation: Total RNA was extracted using ethanol precipitation and TAB-PBiozol. The samples were dissolved in 50 µL of DEPC-treated water, and the total RNA was identified and quantified. Library construction and quality control: mRNA was enriched with a polyA tail and split into small fragments, and the first cDNA was generated by reverse transcription. Second-strand cDNA was synthesized, and end repair and dA-tailing were completed. Sequencing splicing, DNA magnetic bead purification, and fragment selection were performed, and libraries of 250–350 bp inserted fragments were obtained. The linked products were amplified using a polymerase chain reaction, purified using DNA magnetic beads, and dissolved in nuclear-free water. After library construction, concentration and fragment size detection were performed to accurately quantify the effective concentration of the library. Sequencing was performed using the Illumina platform.
Data processing and analysis: The sequencing data were filtered and compared with the Yugu 1 foxtail millet reference genome (http://www.phytozome.net accessed on 11 April 2024) to obtain unique sequence characteristics of the sequenced samples, and differential genes were screened according to gene expression levels for functional annotation and enrichment analysis.

2.6. Statistical Analysis

We used DPS 7.05 for statistical data analysis and Origin 2024 for mapping. A one-way analysis of variance and Duncan’s multiple range test (p < 0.05) were used to test for significance. The results are expressed as mean values ± standard deviation. MetWare Cloud (https://cloud.metware.cn accessed on 11 April 2024) was used to conduct differentially accumulated metabolite (DAM) and differentially expressed gene (DEG) analyses, Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, Gene ontology (GO) classification analysis, the creation of heat maps and orthogonal partial least squares discriminant analysis (OPLS-DA) model score plots, and hierarchical cluster analysis.

3. Results

3.1. Effects of Grain Quality of Foxtail Millet Under Different Treatments

3.1.1. Nutrition Component

Figure 1 shows the impact of different zinc application methods and zinc fertilizer dosages on foxtail millet grain quality. Under zinc fertilizer treatment, the protein and amino acid content in foxtail millet grains showed fluctuating trends, but no significant differences were observed between any treatment and the control (CK). In the basal soil application treatment, the fat content exhibited an increasing trend with increasing application concentration. Conversely, under the foliar spray treatment, the fat content first increased and then decreased. Among these, the F2 treatment had the highest fat content, showing a significant increase of 8.30% compared with CK. The trends for carbohydrate and amylose content were opposite to those for fat content. The F2 treatment had the lowest levels of these components, with significant reductions of 1.43% and 8.82% compared with CK, respectively.

3.1.2. Functional Substance

The contents of total polyphenols, total flavonoids, and yellow pigments in foxtail millet grains were significantly increased by the different Zn fertilizer application methods (Figure 1F–H). The effects of basal application were better than those of foliar spraying, and with an increase in the Zn fertilizer application dose, the content of antioxidant substances in grains first increased and then decreased. Under the S3 and F2 treatments, the contents of total polyphenols, total flavonoids, and yellow pigments were the highest, which were significantly higher than those of CK by 12.44% and 28.10%, 45.87% and 64.40%, and 7.41% and 3.76%, respectively. The response of total flavonoids to the Zn fertilizer was the highest.

3.1.3. Grain Zn Content

As shown in Figure 1I, the Zn content in foxtail millet grains under each treatment was significantly improved compared with CK, and the response of basal Zn fertilizer was greater than that of foliar spraying. With an increase in the application dose, the Zn content of the grains increased. However, under the S3 and S4 treatments, the grain Zn content significantly increased compared with that of CK by 18.97% and 19.46%, respectively. Under the F2 and F3 treatments, the Zn content significantly increased by 26.79% and 30.80%, respectively, compared with that of CK. Figure S1 shows the correlation between grain Zn content and nutritional components. The results showed that fat (p ≤ 0.01), yellow pigment (p ≤ 0.05), and total flavonoids (p ≤ 0.01) were significantly positively correlated with grain Zn content, whereas carbohydrates (p ≤ 0.01) and amino acids (p ≤ 0.01) were significantly negatively correlated with grain Zn content.

3.2. Effects of Foxtail Millet Grain Metabolites Under Different Treatments

3.2.1. Overall Analysis of Metabolites

There were 1584 metabolites identified in foxtail millet grains under the CK, S3, and F2 treatments (Figure S2A). A principal component analysis (PCA) of the above metabolites revealed that PC1 and PC2 contributed 31.65% and 21.90%, respectively. Sample separation between different treatments was obvious, and there was a large difference in the metabolites of foxtail millet grains without spraying and the basal soil application and foliar application of zinc fertilizer (Figure S2B). These identified metabolites were classified into 13 classes, including flavonoids (16.60%), others (14.71%), amino acids and derivatives (12.63%), lipids (11.17%), phenolic acids (10.80%), alkaloids (10.54%), terpenoids (8.27%), lignans and coumarins (4.80%), organic acids (4.80%), nucleotides and derivatives (4.23%), quinones (0.82%), tannins (0.38%), and quinones (0.25%). Flavonoids were the most abundant metabolites detected.

3.2.2. Identification and Analysis of DAMs

After the qualitative and quantitative analysis of the metabolites detected in the samples, differential metabolites were further screened by setting parameters such as a variable importance in projection (VIP) greater than 1, fold change (FC) ≥ 2 or FC ≤ 0.5, and p-value < 0.05 in the OPLS-DA model to find the DAMs in different zinc treatments (Figure 2A). Compared with CK, there were 67 and 48 DAMs in the S3 and F2 treatments, respectively, and 15 DAMs in the two Zn application treatments (Figure 2B). Under the S3 treatment, 35 DAMs were significantly up-regulated compared with CK, while 32 DAMs were significantly down-regulated. Under the F2 treatment, 27 DAMs were significantly up-regulated, and 21 DAMs were down-regulated compared with CK. Additionally, compared with S3, there were 32 DAMs up-regulated and 51 DAMs down-regulated under the F2 treatment (Figure 2C).

3.2.3. KEGG Pathway Analysis of DAMs

We conducted a KEGG pathway analysis to better comprehend the metabolic regulation network of foxtail millet grains following treatment with various zinc fertilizer methods. The significantly enriched 20 pathways of CK vs. S3 and CK vs. F2 are shown in Figure 2D,E. In CK vs. S3, the top six dominant pathways were “biosynthesis of secondary metabolites”, “phenylpropanoid biosynthesis”, “flavone and flavonol biosynthesis”, “tryptophan metabolism”, “nucleotide metabolism”, and “tricin o-glycosides biosynthesis”. In CK vs. F2, “thiamine metabolism”, “carbon metabolism”, “phenylalanine metabolism”, “purine metabolism”, “biosynthesis of cofactors”, and “metabolic pathways” were the top six dominant pathways. Phenylalanine is a synthetic precursor of flavonoids, and metabolic pathways related to flavonoid anabolism have been identified in the treatment of Zn fertilizer spray and base applications. Spraying Zn fertilizer can effectively affect the anabolism of flavonoids. Significant changes in flavonoid biosynthesis have also been found in studies on Zn fertilizer spraying in plants, such as Acanthopanax senticosus [28].

3.2.4. Flavonoid DAMs

Based on metabolomic studies, Utasee et al. [29] have suggested that after the application of Zn, the anthocyanin content in purple rice improved, and Wang et al. [30] have shown that Zn fertilizer increased the proline (Pro), asparagine (Asp), citric acid, and malic acid content in rice. We classified DAMs into 10 categories: alkaloids, amino acids and derivatives, flavonoids, lipids, nucleotides and derivatives, organic acids, others, phenolic acids, terpenoids, and lignans and coumarins (Figure 2F). Flavonoids are the most identified DAMs [31]. Figure 2G shows the changes in flavonoid DAMs under different Zn fertilizer treatments. There were 19 flavonoids DAMs between CK and S3 and 7 flavonoid DAMs between CK and F2. Under the S3 treatment, a total of 11 flavonoid DAMs were significantly down-regulated compared with CK, while 8 DAMs were significantly up-regulated compared with CK. However, compared with CK, only one flavonoid DAM was significantly up-regulated, and six DAMs were significantly down-regulated under the F2 treatment. Obviously, different zinc fertilizer treatments have significant effects on the content of flavonoid DAMs. The effects of the basal soil application of zinc fertilizer on the types and contents of flavonoid metabolites in the grains were significantly greater than those of foliar application.

3.3. Effects of Grain Transcriptome of Foxtail Millet Under Different Treatments

3.3.1. Overview of Transcriptome Data

The transcriptome sequencing (RNA-seq) results are shown in Figure S2A. The percentage of Q30 bases in each sample was >94.29, indicating high-quality sequencing data (Table S4). Table S5 compares the sequencing data with the reference genome. The ratio of sequencing reads produced in this experiment to the genome was >70%, indicating that the test results were satisfactory and that there was no contamination during the test process. To better understand the correlations between treatments, we performed PCA on the entire dataset, which visually displayed the transcriptional characteristics (Figure S3B). The transcriptome sequencing data were annotated to the database, of which 26458 genes were annotated to the GO database, and 18435 genes were annotated to the KEGG database.

3.3.2. Identification and Analysis of DEGs

The DEGs of foxtail millet grains treated with different zinc application methods were screened. A total of 493 DEGs were identified, and gene expression levels were compared among the three treatments. To determine distinct DEGs for each comparison, a Venn diagram was created. A comparison of DEGs across the three sets revealed 287, 53, and 72 unique DEGs in the S3 vs. CK, F2 vs. CK, and F2 vs. S3 comparison sets, respectively (Figure S3C). In addition, there were 34 common DEGs between S3 vs. CK and F2 vs. CK. As shown in Figure 3A,B, there were 240 DEGs up-regulated and 124 DEGs down-regulated under the S3 treatment compared with CK. Under the F2 treatment, 88 and 3 DEGs were up-regulated and down-regulated, respectively, compared with CK. In addition, compared with S3, there were 72 DEGs up-regulated and 47 DEGs down-regulated under the F2 treatment. Therefore, both foliar and soil-based applications significantly affected gene expression in grain.

3.3.3. GO Classification Analysis and KEGG Pathway Analysis of DEGs

A GO classification study of the unique DEGs found in the S3 vs. CK and F2 vs. CK samples was performed to further analyze the DEGs (Figure 3C,D). The DEGs were divided into the biological process (BP), cellular component (CC), and molecular function (MF) categories. In S3 vs. CK, cellular anatomical entity, binding, catalytic activity, cellular process, and metabolic process were the top five classes. Cellular anatomical entity, cellular process, and metabolic process were the top three classes in the F2 vs. CK comparison. The impact of Zn fertilizer spraying on foxtail millet DEGs was smaller than that of the soil-based application. When subjected to a KEGG pathway analysis, 20 significantly enriched pathways of CK vs. S3 and CK vs. F2 were identified, as shown in Figure 3E,F. In CK vs. S3, “photosynthesis”, “starch and sucrose metabolism”, “metabolic pathways”, “plant hormone signal transduction”, and “biosynthesis of secondary metabolism” were the top five dominant pathways. Meanwhile, the top five dominant pathways in CK vs. F2 were “photosynthesis”, “metabolic pathways”, “circadian rhythm-plant”, “carotenoid biosynthesis”, and “biosynthesis of secondary metabolites”. In addition, we found pathways related to flavonoid anabolism in the KEGG pathway analysis of the DEGs.

3.3.4. Flavonoid DEGs

Han et al. [32] have found that the CHIL, CHSC2, ANR2, PAL1, PAL2, PAL5, and PAL7 expression levels in foxtail millet varieties with high flavonoid content were relatively high, and these may be the key genes that determine the change in flavonoid content in foxtail millet panicles. In the present study, seven DEGs were identified and found to be associated with the biosynthesis and metabolism of flavonoids in CK vs. S3 (Table 1). Three DEGs were annotated as being involved in the flavonoid metabolic and biosynthetic process: one was identified as a chalcone isomerase, two as flavanone-3-hydroxylase, and one as a leucoanthocyanidin reductase. There were two DEGs identified that are associated with the biosynthesis and metabolism of flavonoids in CK vs. F2. One DEG was annotated as being involved in a flavonoid metabolic and biosynthetic process, and the other was annotated as chalcone isomerase. The levels of flavonoid DEGs were significantly higher in both the soil-based application and foliar application of Zn fertilizer than in CK.

3.4. Flavonoid Biosynthesis Pathway

Phenylalanine is the initial precursor of flavonoid biosynthesis, and phenylalanine ammonia-lyase (PAL) converts it into cinnamic acid. Cinnamic acid is then converted into coumaroyl-CoA by cinnamate-4-hydroxylase (C4H) and 4-coumarate-CoA ligase (4CL). Chalcone synthase (CHS) catalyzes the conversion of coumaroyl-CoA to chalcone. The chalcones are then transformed into flavanones by chalcone isomerase (CHI). Flavanone is further modified by flavanone-3-hydroxylase (F3H) to form dihydrokaempferol, or it can be converted into flavonoids such as luteolin, apigenin, and vitexin by flavonoid synthase (FNS). Additionally, flavanones can be catalyzed by isoflavone synthase (IFS) to produce isoflavones. Dihydrokaempferol is transformed into kaempferol by flavonol synthase (FLS), which can also be converted into flavanols by dihydroflavonol reductase (DFR) and leucoanthocyanidin reductase (LAR). Finally, flavanols are further processed by anthocyanidin synthase (ANS) and UDP-glucose flavonoid glucosyltransferase (UFGT) to produce anthocyanins [33]. In this pathway, we identified two differentially expressed genes, Seita.4G186000 and Seita.9G0.4700, that are involved in the regulation of C4H synthesis. Compared with CK, both S3 and F2 showed up-regulated expression levels. Additionally, we identified Seita.9G561700 and Seita.9G561500 as participants in the regulation of F3H synthesis, with S3 and F2 exhibiting up-regulated expression relative to CK. Seita.2071700 is involved in the regulation of LAR synthesis, and its expression levels in S3 and F2 were also up-regulated compared with those in CK (Figure 4).

4. Discussion

Proteins, fats, and carbohydrates are collectively referred to as the three major nutrients. Previous research has shown that there was no significant difference in the fat and protein content of wheat grains between the foliar application of ZnSO4 and CK [8,34]. In this study, we found that the protein and amino acid contents in foxtail millet did not show significant changes with the application of zinc fertilizer, indicating that the use of zinc fertilizer did not lead to nutrient loss in foxtail millet. Yu et al. [20] have suggested that Zn fertilizer application does not significantly affect the starch content of maize grains. However, zinc treatment can decrease rice amylose content [35]. Our study demonstrated that foliar spraying with zinc fertilizer significantly reduced the amylose content in foxtail millet, whereas basal soil application had no significant effect. The responses of different crops to Zn fertilizer application differed, and the fertilization method affected the amylose content of the grains. Polyphenols, flavonoids, and flavones are characteristic antioxidant substances in foxtail millet that can clear free radicals and enhance immune function in humans [27,36]. In this study, the content of total polyphenols, total flavonoids, and yellow pigments in foxtail millet grains significantly increased after Zn fertilizer application, and the effects of foliar spraying were better than those of basal application. This is mainly due to the fact that foliar spraying directly transports Zn to the main part of flavonoid synthesis—the leaves. At the grain filling stage, zinc maximizes the activation of key enzymes in the flavonoid biosynthesis pathway, thereby enhancing the synthesis capacity of the source organ, promoting more flavonoid synthesis and transport to the sink organ (grain), and ultimately leading to a significant increase in the grain flavonoid content. Mahasweta et al. [37] have also demonstrated that different zinc application methods have different effects on the accumulation of nutrient quality in cereals. According to Zhao et al. [38], the zinc content of wheat grains was significantly improved in the foliar spray treatment compared with that in CK, but there was no significant difference between the basal Zn fertilizer treatment and CK. The insufficient dietary intake of Zn is an important health problem; therefore, it is important to increase the Zn content in crop grains through Zn biofortification [39]. Our study indicated that zinc fertilizer could enhance the Zn content of foxtail millet grains and improve the nutritional value of foxtail millet.
In the analysis of foxtail millet grain quality, we found that the S3 (basal soil application) and F2 (foliar application) treatments demonstrated optimal comprehensive enhancement within their respective fertilization methods. The content of functional compounds, particularly total polyphenols, total flavonoids, and yellow pigments, was significantly higher in the two treatments than in the other treatments. S3 and F2 were selected for the subsequent transcriptomic and metabolomic studies. Integrated transcriptomic and metabolomic analyses revealed that zinc fertilizer application methods significantly influenced the molecular mechanisms and accumulation efficiency of flavonoid biosynthesis in foxtail millet. The basal soil application of zinc fertilizer activates a multi-level gene network governing flavonoid synthesis through the dynamic processes of root absorption, xylem transport, and grain accumulation. Zinc ions are absorbed by roots via ZIP transporters, transported through the xylem mediated by HMA family proteins, compartmentalized into vacuoles within cells by MTP transporters, and stored chelated with PCs [11]. This continuous slow uptake process acting throughout the entire growth period significantly up-regulated seven flavonoid-related DEGs, covering both the upstream (C4H: Seita.4G186000) and downstream nodes (F3H: Seita.9G561700/Seita.9G561500; LAR: Seita.2G071700) of the phenylpropanoid pathway. Among these, C4H, a rate-limiting enzyme in the phenylpropanoid pathway, is up-regulated (Seita.4G186000), directly promoting the synthesis of the flavonoid precursor coumaroyl-CoA [31]. Furthermore, soil basal zinc application induced changes in 19 flavonoid DAMs (11 up-regulated and 8 down-regulated). However, the overall increase in the total flavonoid content (45.87%) was lower than that achieved by foliar spraying. This apparent paradox may stem from metabolic partitioning between tissues and organs, which is regulated by source–sink relationships. While upstream gene activation increased the synthesis of precursors such as naringenin, downstream glycosyltransferases (such as UFGT) were not synchronously up-regulated, limiting the stable accumulation of end products [9]. Concurrently, oxidative degradation intensified, and the continuous zinc uptake may elevate reactive oxygen species levels, accelerating flavonoid degradation [31]. Foliar spraying with zinc fertilizer enables targeted and efficient regulation through the absorption pathway via the cuticle/stomata followed by phloem retranslocation. After penetrating the leaf, zinc is transported intracellularly by ZIP transporters, with a portion rapidly translocated to the grains via the phloem [11]. During the grain-filling stage in foxtail millet, the leaves rapidly absorb Zn fertilizer, precisely regulating the core synthesis nodes. Only, two flavonoid-related DEGs were significantly up-regulated. Among these, C4H (Seita.4G186000) directly strengthened the supply of flavonoid precursors, whereas the gene associated with flavonoid metabolism and biosynthesis (Seita.2G324600) coordinated the metabolic flux. Compared with basal soil application, foliar spraying did not activate the downstream genes F3H and LAR, indicating that foliar spraying drives synthesis primarily by enhancing upstream flux. Under foliar spraying, the increase in total flavonoid content in foxtail millet grains reached 64.40%, which was significantly higher than that achieved with basal soil application. Only seven flavonoid DAMs were altered (one up-regulated, six down-regulated), demonstrating that minor genetic regulations can substantially enhance the end product. This is attributed to the alleviation of the rate-limiting constraint imposed by C4H [16]. Concurrently, the rapid uptake of zinc may minimize the reactive oxygen species exposure time and reduce the oxidative degradation of flavonoids [27].

5. Conclusions

Foliar spraying and the basal soil application of Zn fertilizer significantly improved the carotenoid, total polyphenol, total flavonoid, and Zn contents in foxtail millet grains, and the changes in the S3 and F2 treatments were the most significant. The basal soil application of Zn increased the flavonoid content of foxtail millet by up-regulating flavonoid metabolic and biosynthetic processes, flavanone-3-hydroxylase, chalcone isomerase, and leucoanthocyanidin reductase-related genes. Foliar Zn fertilization increased the flavonoid content of foxtail millet by up-regulating flavonoid metabolic and biosynthetic processes and chalcone isomerase-related genes. The foliar application of Zn fertilizer has a more efficient biofortification effect on foxtail millet than basal soil application. However, this experiment was only a single-season study and has certain limitations; future studies should involve multiple seasons.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15081767/s1, Figure S1: Correlation analysis among the nutritional qualities of foxtail millet grains, where *, **, and *** indicate significance at 0.05, 0.01, and 0.001, respectively. Figure S2: Heat map analysis, principal component analysis (PCA), and composition classification of foxtail millet metabolites under different Zn fertilizer application methods. (A) Clustering heat map of all metabolites; (B) 2D PCA Plot; Figure S3: Heat map analysis, principal component analysis (PCA), and Venn diagram of foxtail millet DEGs under different Zn fertilizer application methods. (A) Clustering heat map of all DEGs. (B) PCA score plot of different treatments. (C) Venn diagram of overlapping and unique DEGs amongst different treatments; Table S1: The basic properties of the test field; Table S2: Experimental treatments; Table S3: Chromatographic conditions of metabolome analysis; Table S4: Transcriptome sequencing data statistics and quality assessment; Table S5: Statistics of comparison of sequencing results.

Author Contributions

K.M.: Investigation, Data curation, Formal analysis, and Writing—original draft. X.L.: Investigation, Data curation, and Formal analysis. X.C.: Investigation, Data curation, and Formal analysis. C.W.: Investigation, Data curation, and Formal analysis. Z.Z.: Investigation, Data curation, and Formal analysis. X.Y.: Conceptualization and Writing—review and editing. F.C.: Conceptualization, Funding acquisition, and Writing—review and editing. X.W.: Conceptualization and Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Key Research and Development Program of China (2021YFD1901103). We are grateful to the anonymous reviewers for their insightful comments and input.

Data Availability Statement

The data will be made available upon reasonable request.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Grain quality of foxtail millet under different Zn fertilizer application methods and concentrations. Nutrition component indicators include protein (PC, (A)), fat (FC, (B)), carbohydrate (CC, (C)), amino acid (AAC, (D)), and amylose (AC, (E)). Functional substance indicators include total polyphenols (TPC, (F)), yellow pigment (YPC, (G)), and total flavonoids (TFC, (H)). Mineral element indicators include Zn (ZnC, (I)). Data are presented as means ± standard deviations. Different letters indicate significant differences at p ≤ 0.05 among different treatments. S means basal soil application, and F means foliar application.
Figure 1. Grain quality of foxtail millet under different Zn fertilizer application methods and concentrations. Nutrition component indicators include protein (PC, (A)), fat (FC, (B)), carbohydrate (CC, (C)), amino acid (AAC, (D)), and amylose (AC, (E)). Functional substance indicators include total polyphenols (TPC, (F)), yellow pigment (YPC, (G)), and total flavonoids (TFC, (H)). Mineral element indicators include Zn (ZnC, (I)). Data are presented as means ± standard deviations. Different letters indicate significant differences at p ≤ 0.05 among different treatments. S means basal soil application, and F means foliar application.
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Figure 2. Grain metabolites of foxtail millet under different Zn fertilizer application methods. (A) OPLS-DA model score plots of foxtail millet under different treatments. (B) Venn diagram of overlapping and unique metabolites among different treatments. (C) Number of up- and down-regulated differentially accumulated metabolites (DAMs) in different treatments. (D,E) Top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DAMs in different treatments (CK vs. S3/F2). (F) Composition classification of DAMs under different treatments. (G) Changes in flavonoid DAMs under different treatments.
Figure 2. Grain metabolites of foxtail millet under different Zn fertilizer application methods. (A) OPLS-DA model score plots of foxtail millet under different treatments. (B) Venn diagram of overlapping and unique metabolites among different treatments. (C) Number of up- and down-regulated differentially accumulated metabolites (DAMs) in different treatments. (D,E) Top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of DAMs in different treatments (CK vs. S3/F2). (F) Composition classification of DAMs under different treatments. (G) Changes in flavonoid DAMs under different treatments.
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Figure 3. Grain transcriptome of foxtail millet under different Zn fertilizer application methods. (A,B) Number of up- and down-regulated differentially accumulated metabolite differentially expressed genes (DEGs) in different treatments. (C,D) Gene ontology (GO) classification analysis of DEGs in different treatments (CK vs. S3/F2). (E,F) KEGG pathways of DEGs in different treatments (CK vs. S3/F2).
Figure 3. Grain transcriptome of foxtail millet under different Zn fertilizer application methods. (A,B) Number of up- and down-regulated differentially accumulated metabolite differentially expressed genes (DEGs) in different treatments. (C,D) Gene ontology (GO) classification analysis of DEGs in different treatments (CK vs. S3/F2). (E,F) KEGG pathways of DEGs in different treatments (CK vs. S3/F2).
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Figure 4. The pathway of differential flavonoid metabolites. Black letters represent flavonoid metabolites. Orange letters represent enzymes in metabolic pathways. The heat maps show the expression level of DEGs. As shown in the upper-right bar, green indicates a lower expression level, whereas red indicates a higher expression level.
Figure 4. The pathway of differential flavonoid metabolites. Black letters represent flavonoid metabolites. Orange letters represent enzymes in metabolic pathways. The heat maps show the expression level of DEGs. As shown in the upper-right bar, green indicates a lower expression level, whereas red indicates a higher expression level.
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Table 1. Differential gene information related to flavonoid synthesis.
Table 1. Differential gene information related to flavonoid synthesis.
Gene IDFunction AnnotationS3 vs. CKF2 vs. CK
Seita.2G324600.v2.2Flavonoid metabolic and biosynthetic processupup
Seita.4G186000.v2.2Chalcone isomerase up
Seita.9G561700.v2.2Flavanone-3-hydroxylaseup
Seita.2G289600.v2.2Flavonoid metabolic and biosynthetic processup
Seita.9G034700.v2.2Chalcone isomeraseup
Seita.9G561500.v2.2Flavanone-3-hydroxylaseup
Seita.9G142900.v2.2Flavonoid metabolic and biosynthetic processup
Seita.2G071700.v2.2Leucoanthocyanidin reductaseup
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Ma, K.; Li, X.; Chen, X.; Wang, C.; Zhang, Z.; Yuan, X.; Chen, F.; Wen, X. Transcriptome and Metabolome Revealed Impacts of Zn Fertilizer Application on Flavonoid Biosynthesis in Foxtail Millet. Agronomy 2025, 15, 1767. https://doi.org/10.3390/agronomy15081767

AMA Style

Ma K, Li X, Chen X, Wang C, Zhang Z, Yuan X, Chen F, Wen X. Transcriptome and Metabolome Revealed Impacts of Zn Fertilizer Application on Flavonoid Biosynthesis in Foxtail Millet. Agronomy. 2025; 15(8):1767. https://doi.org/10.3390/agronomy15081767

Chicago/Turabian Style

Ma, Ke, Xiangyu Li, Xiangyang Chen, Chu Wang, Zecheng Zhang, Xiangyang Yuan, Fu Chen, and Xinya Wen. 2025. "Transcriptome and Metabolome Revealed Impacts of Zn Fertilizer Application on Flavonoid Biosynthesis in Foxtail Millet" Agronomy 15, no. 8: 1767. https://doi.org/10.3390/agronomy15081767

APA Style

Ma, K., Li, X., Chen, X., Wang, C., Zhang, Z., Yuan, X., Chen, F., & Wen, X. (2025). Transcriptome and Metabolome Revealed Impacts of Zn Fertilizer Application on Flavonoid Biosynthesis in Foxtail Millet. Agronomy, 15(8), 1767. https://doi.org/10.3390/agronomy15081767

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