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Article

Transcriptome and Metabolome Analysis of Organic and Chemical Fertilizer Effects on Highland Barley Growth and Nutrient Utilization

by
Jiahui Yan
,
Hua Weng
,
Lu Hou
,
Qiang Yao
and
Tao He
*
State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(2), 380; https://doi.org/10.3390/agronomy15020380
Submission received: 9 December 2024 / Revised: 20 January 2025 / Accepted: 28 January 2025 / Published: 31 January 2025
(This article belongs to the Section Soil and Plant Nutrition)
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Abstract

:
The rate of chemical fertilizers’ utilization by crops does not exceed 40%. Environmental pollution and resource waste caused by the excessive application of chemical fertilizers have led to increased interested in using organic fertilizers as replacements for chemical ones. The present study investigated the impact of the combined application of chemical and organic fertilizers on the growth and nutrient utilization efficiency of highland barley (Hordeum vulgare L.). Compared to the application of 100% chemical fertilizers (OFR0), the substitution of chemical fertilizer with 40% organic fertilizer (OFR40) resulted in a significant increase in root length by 4% and improved the nutrient absorption capacity. The crop yields at harvest were not diminished in the combined fertilizer group compared to the OFR0 treatment group, while simultaneously achieving a 60% reduction in chemical fertilizer application. However, a complete substitution with 100% organic fertilizer (OFR100) resulted in a lower yield. This suggests that appropriate proportions of organic fertilizer replacement can maintain yield by increasing root length and enhancing the crop’s nutrient absorption capacity. In order to elucidate the mechanisms by which organic fertilizer modulates crop growth and nutrient utilization efficiency, combined transcriptomic and metabolomic analysis revealed that as the concentration of organic fertilizer increased, the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) shifted from secondary metabolite synthesis toward nitrogen metabolism. In addition, the gene expression and enzymatic activity of NR (nitrate reductase), GS (glutamine synthetase), and GOGAT (glutamine oxoglutarate aminotransferase) (key genes in the nitrogen metabolism pathways) were significantly enhanced in the OFR40 group. This study’s omics-based approach demonstrates that the combined use of chemical and organic fertilizers enhances nitrogen absorption and utilization through an increased expression of key genes and enzymatic activities within the nitrogen metabolic pathways. This synergistic effect not only maintains crop yields but also reduces the reliance on chemical fertilizers, offering a sustainable strategy for agricultural production.

1. Introduction

The application of fertilizers is crucial for agricultural production. In cereal crops, fertilizer availability influences yield, nitrogen uptake efficiency, and grain protein content [1]. The increase in global grain and fruit yields in recent decades has been largely due to the use of fertilizers [2]. However, the excessive use of chemical fertilizer aggravates soil acidification, hardening, and nutrient loss, leading to declines in crop yield [3]. Previous studies have indicated that plants do not absorb all of the millions of tons of synthetic nutrients applied to the soil annually [4]. For example, excessive amounts of nitrogen (N) and phosphorus (P) applied to agricultural soils have been reported to leach from fields into the atmosphere or nearby water sources, generating greenhouse gases and causing eutrophication in aquatic systems and soil salinization [4]. Furthermore, the excessive application of chemical fertilizers has led to declining food safety and quality [5,6]. Conversely, the application of organic fertilizers, which are nutrient-rich and exhibit a slow nutrient release rate, can help to alleviate the degradation of soil physicochemical and biological properties often associated with the use of chemical fertilizers. Organic fertilizers also supply a variety of nutrients for crop growth, stimulate microbial reproduction, and improve the soil’s water and nutrient retention capabilities [7]. Previous studies have shown that the combined application of organic and chemical fertilizers significantly improves soil fertility and bacterial community composition [8], enhances soil microbial reproduction, and increases crop yield [9]. Therefore, the use of organic fertilizers can mitigate the adverse effects of chemical fertilizer application in agricultural systems.
The use of organic fertilizers has been shown to increase production stability while reducing chemical fertilizer usage and its related environmental hazards [10]. In rice, the combined application of manure and synthetic N fertilizers improved yield by about 15% [11]. Another study showed that inorganic fertilizer enhanced the number of fruits and the weight of the fruits in tomato and cucumber plants compared to organic fertilizer [12]. Studies have demonstrated that bio-organic fertilizers alter soil chemical properties and modulate specific microbial taxa and functions, promoting increased yield in pear plants [13]. The presence of bio-organic fertilizer increased the yield of Chinese baby cabbage by enhancing the physicochemical properties of soil and improving the abundance and diversity of beneficial bacteria and fungi [14]. Therefore, existing research has confirmed that organic fertilizers increase crop yield by improving the abundance and diversity of soil microorganisms. Understanding the effect of organic fertilizers on plant yield and carbon and nitrogen utilization is crucial for sustainable agriculture.
Highland barley (Hordeum vulgare L.) is the fourth most produced cereal globally and is mainly distributed in highland areas worldwide, including China. It is one of the most important cereal crops on the Qinghai–Tibet Plateau, which is the highest altitude plateau in the world and the largest in China [15]. Highland barley contains various nutrients, such as bioactive carbohydrates, polyphenols, minerals, vitamins, phenolic acid, flavonoids, and β-glucan. Furthermore, barley is gaining popularity due to its high protein, fiber, and vitamin content; low fat and sugar levels; and beneficial functional components [16]. Compared with conventional barley varieties, highland barley has higher protein, higher dietary fiber, and lower fat [17], which meet the preferences of modern people. The cultivation of highland barley faces the challenges of poor soil quality and vulnerability to climate fluctuations, making it prone to problems such as drought, cold stress, and hail damage. Therefore, because of its weaker ability to resist natural disasters, highland barley typically exhibits low yields, often failing to meet planting expectations. Recent research has found that a balanced fertilization regimen can effectively address these issues. Furthermore, it is crucial to investigate how to optimize the yield and quality of highland barley through appropriate fertilization practices. The mechanisms of nutrient uptake and utilization in plants under different fertilization methods also require investigation. Understanding these processes is essential for developing effective agricultural strategies that enhance crop performance while ensuring sustainable soil health.
Considering all these points, the current study analyzed the impacts of different fertilization schemes on the growth and development of highland barley, analyzed the mechanism by which organic fertilizer regulates plant growth and development, and provides effective methods for efficient fertilization. This study contributes to the understanding of the patterns and mechanisms of nutrient uptake and utilization in plants as influenced by fertilizer treatments and also provides techniques for the rational and sustainable use of fertilizers.

2. Materials and Methods

2.1. Plant Materials and Experimental Design

Highland barley variety Kunlun 14 was used in this study. The seeds of this variety were collected from Xining City, Qinghai Province, China (101°74′ E, 36°56′ N, H: 2261 m), and deposited at the National duplicate GenBank for crops (Qinghai Academy of Agriculture and Forestry Sciences), Xining City. The soil tested was matrix soil collected from the Experimental Station of the Qinghai Academy of Agricultural and Forestry Sciences (101°74′ E, 36°56′ N, H: 2261 m). The soil was air-dried and sieved through a 2 mm mesh. The soil type is classified as calcic chestnut soil, and its basic physicochemical properties are as follows: 25.03 g·kg−1 organic matter content, 1.36 g·kg−1 total nitrogen, 12.42 mg·kg−1 nitrate nitrogen, 5.37 mg·kg−1 ammonium nitrogen, 8.22 pH, 144.0 mg·kg−1 available nitrogen, 136.0 mg·kg−1 available potassium, and 28.8 mg·kg−1 available phosphorus. Stanley commercial organic fertilizer (N:P2O5:K2O = 5.89:1.12:0.81; organic matter content = 52.33%) and chemical fertilizers, including urea (N = 46%), potassium sulfate (K2O = 12%), and superphosphate calcium (P2O5 = 12%), were used in the study. All treatments were applied with the same nutrient standard of N 164.5 mg·kg−1, P2O5 185.5 mg·kg−1, and K2O 70 mg·kg−1, and the corresponding contents are listed in Table 1.
Highland barley seedlings were maintained under day/night temperatures of 12.5 ± 1.5/22.0 ± 2.0 °C and a relative humidity of 60.0 ± 5.1%, 12 h light/12 h dark [18]. Three fertilizer treatments were applied to the barley seedlings: OFR0, with 0% organic fertilizer substitution; OFR40, with 40% organic fertilizer substitution; and OFR100, with 100% organic fertilizer substitution. The nitrogen fertilizer dosage was consistent for all treatments. The seedlings were grown in pots with 3.5 kg of soil mixed with all fertilizers; 40 pots were maintained per treatment, and 120 pots were used per experiment. Ten pots were randomly selected from each treatment at the seedling, tillering, heading, and flowering stages to analyze the plant growth parameters, carbon and nitrogen content, and nitrogen metabolic enzyme activity.

2.2. Study of Plant Growth and Root Morphology

The aboveground and belowground parts of highland barley plants grown under the OFR0, OFR40, and OFR100 treatments were collected at four growth stages (seedling stage, jointing stage, heading stage, and flowering stage). The root was cleaned using water, and excess water was blotted dry with filter paper. The height (cm) and root length (cm) were measured using a ruler. The fresh shoots and roots were weighed, placed in an oven at 120 °C for 30 min, and then dried at 80 °C until reaching a constant weight [19]. The dry weights were recorded.

2.3. Determination of Nutrient Uptake by Plants

The plant uptake of nutrients, including nitrogen (N), phosphorus (P), and potassium (K), was determined. Nitrogen uptake was determined by semimicro Kjeldahl (KDY-9820) digestion [20]. Phosphorus was determined colorimetrically using the molybdate method [21]. Potassium was measured via flame spectrophotometry. The C/N ratio was determined following the method described by Moreno-Pedraza [22].

2.4. RNA Extraction and Sequencing

Leaf and root samples were harvested from plants under OFR0, OFR40, or OFR100 treatments and stored at −80 °C for metabolite profiling and RNA sequencing. Total RNA was isolated from the leaf samples using a Sangon RNA extraction kit (Shanghai, China, https://store.sangon.com/productDetail?productInfo.code=B518631#). The mRNA was purified, and the library was prepared for transcriptome sequencing, as described previously [18,23].

2.5. Transcriptome Analysis

The RNA libraries were sequenced on an Illumina HiSeq X Ten platform to generate 150 bp paired-end reads. The raw data in fastq format were first processed using Trimmomatic to remove low-quality reads. The raw count data were acquired using Counts and Rscript software features. The clean reads were mapped to the highland barley reference genome using HISAT2 [24]. With the Trinity software, gene expression was estimated based on transcripts per million (TPM). Furthermore, based on the raw count matrix, differentially expressed gene (DEG) analyses were carried out with the DESeq2 method using the edgeR (3.22.5) package in R [25].

2.6. Metabolite Extraction and UHPLC-MS/MS Analysis

The metabolites were extracted and analyzed through ultra-high-performance liquid–tandem mass spectrometry (UHPLC-MS/MS), as reported in [18]. Approximately 100 mg of tissue was ground with liquid nitrogen and resuspended in prechilled 80% methanol and 0.1% formic acid by vortexing. The sample was then incubated on ice for 5 min and centrifuged at 15,000 rpm and 4 °C for 5 min. A portion of the supernatant was diluted to a final concentration of 53% methanol with LC-MS-grade water, transferred to a fresh Eppendorf tube, and centrifuged at 15,000 g and 4 °C for 10 min. Finally, the supernatant was injected into an LC-MS/MS system for analysis. The liquid sample (100 μL) and prechilled methanol (400 μL) were mixed by vertexing, and then mixed with prechilled 80% methanol by vortexing, followed by sonication for 6 min [18]. UHPLC-MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher, Bremen, Germany) coupled with an Orbitrap Q ExactiveTM HF mass spectrometer (Thermo Fisher, Bremen, Germany) at Novogene Co., Ltd. (Beijing, China).

2.7. Combined Transcriptome and Metabolome Analyses

Furthermore, the DEGs and differentially accumulated metabolites (DAMs) were analyzed in an integrated manner to determine the degree of enrichment of metabolic pathways. Gene–metabolite networks with a Pearson correlation coefficient (PCC) > 0.8 were used to construct the transcript–metabolite network [26].

2.8. Assessment of Enzymatic Activities in Nitrogen Metabolism

Barley was harvested at four growth stages for the determination of nitrogen metabolism-related enzyme activities. Enzyme activities of glutamate pyruvate transaminase (GPT, http://www.cominbio.com/a/shijihe/shenghuashiji/anjisuandaixiexilie/2014/1112/470.html (accessed on 16 January 2025)), glutamine oxoglutarate aminotransferase (GOGAT, http://www.cominbio.com/a/shijihe/shenghuashiji/dandaixiexilie/2014/1112/463.html (accessed on 16 January 2025)), glutamate oxaloacetate transaminase (GOT, http://www.cominbio.com/a/shijihe/shenghuashiji/anjisuandaixiexilie/2014/1112/471.html (accessed on 16 January 2025)), nitrate reductase (NR, http://www.cominbio.com/a/shijihe/shenghuashiji/dandaixiexilie/2020/0519/1938.html (accessed on 16 January 2025)), and glutamine synthetase (GS, http://www.cominbio.com/a/shijihe/shenghuashiji/anjisuandaixiexilie/2014/1112/472.html (accessed on 16 January 2025)) were determined using kits from Suzhou Koming Biotechnology Co., Ltd. Enzyme activities were calculated per unit of fresh weight of the samples. Specifically, GOT and GPT were defined as one activity unit per gram of tissue per minute, catalyzing the production of 1 nmol of pyruvate [27]. GOGAT was defined as one activity unit per gram of tissue per minute, consuming 1 nmol of NADH [28]. NR was defined as one activity unit per gram of fresh weight sample, catalyzing the reduction of 1 nmol of NADH per minute [29]. GS was defined as one activity unit per gram of tissue, producing 1 μmol of γ-glutamyl hydroxamate per hour per milliliter of reaction system [29].

2.9. Data Analysis

Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed with MetaX (http://metax.genomics.cn). The DAMs were selected based on the variable influence on projection (VIP) values obtained from the OPLS-DA model and p-value; metabolites with VIP > 1.0 and p-value < 0.05 were considered DAMs [30]. Furthermore, the KEGG database was employed to determine high-level functions of DAMs [31]. All the experiments described in this paper were carried out on three independent samples to ensure that the trends and relationships observed in the cultures were reproducible. Finally, univariate analysis (t-test) was performed to determine the statistical significance (p-value).

3. Results

3.1. Growth and Yield

The present study tested the effects of three fertilization schemes on plant growth for application at different growth stages (seedling stage, jointing stage, heading stage, and flowering stage) in highland barley: chemical fertilizer (OFR0), combination of chemical fertilizer and organic fertilizer (OFR40), and use of organic fertilizer (OFR100). The effects of different fertilization schemes on highland barley yield were also tested. The plant height, aboveground fresh weight (UP FW), and aboveground dry weight (UP DW) of highland barley significantly decreased as the concentration of organic fertilizer increased at four growth stages. This study found that plant height, aboveground fresh weight, and aboveground dry weight were highest when 100% chemical fertilizer (OFR0) was applied, while the lowest values for plant height were observed with 100% organic fertilizer (OFR100) (Figure 1A). The application of 100% organic fertilizer (OFR100) resulted in the highest values of root length, underground fresh weight (under FW), and underground dry weight (under DW) at different growth stages, while the lowest values were found with the use of 100% chemical fertilizer (OFR0) (Figure 1A). These results indicate that 100% chemical fertilizer promotes plant height, while 100% organic fertilizer significantly increases root length. The highland barley yield, including the number of grains per spike, 1000-grain weight, and yield per plant, significantly decreased with increasing concentration of organic fertilizer. The lowest values of highland barley yield were observed under OFR100 treatments. No significant differences were observed in the highland barley yield between OFR0 and OFR40 (Figure 1B). The results indicate that the application of 40% organic fertilizer as a substitute for chemical fertilizer did not result in any significant decreases in crop productivity while simultaneously achieving a 60% reduction in chemical fertilizer application. The combination of chemical fertilizer and organic fertilizer is a potential recommendation for reducing chemical fertilizer use.

3.2. Nutrient Uptake

To investigate the effects of different fertilization treatments on the nutrient absorption of highland barley, the contents of nitrogen, phosphorus, and potassium were detected in leaves and root tissues. Compared with the chemical fertilizer treatment, organic fertilizer significantly decreased the nitrogen content in leaves. The changing trends in phosphorus and potassium content was consistent with nitrogen content after organic fertilizer treatment. The contents of nitrogen, phosphorus, and potassium were highest when 100% chemical fertilizer was applied, while the lowest values for nitrogen, phosphorus, and potassium content were observed with 100% organic fertilizer in the leaves (Figure 2A–C). The substitution of chemical fertilizer with organic fertilizer treatments significantly improved the nitrogen, phosphorus, and potassium contents in the root tissues. The contents of nitrogen, phosphorus, and potassium were highest in the 100% organic fertilizer treatment, while the lowest values for nitrogen, phosphorus, and potassium were observed in the roots of plants receiving 100% chemical fertilizer (Figure 2A–C). The results indicate that the use of organic fertilizer significantly improved nutrient uptake in the root compared to traditional chemical fertilizer treatments. However, organic fertilizer alone was not conducive to nutrient absorption in the leaves.

3.3. C/N Ratio

The C/N ratio in plant tissues is indicative of growth status and nutrient absorption capacity. The C/N ratio was assessed in both the leaves and roots of highland barley in this study. The results suggest that a significant increase could be observed in the carbon/nitrogen (C/N) ratio in the leaves with the increase in organic fertilizer concentration. The application of the OFR100 treatment resulted in the highest C/N ratio in the leaves, while the lowest ratio was found with the use of the OFR0 treatment (Figure 3A). The substitution of chemical fertilizer with organic fertilizer significantly decreased the C/N ratio in the root tissues. The lowest C/N ratio in the roots was found with the use of the OFR100 treatment, while the highest values of the C/N ratio were found with the use of the OFR0 treatment (Figure 3B). As the concentration of organic fertilizer increases, the C/N ratio in the leaves increases, which may be due to slower nutrient absorption and weakened nitrogen utilization in the leaves. The decrease in the C/N ratio in roots may be due to enhanced nutrient absorption and accelerated nitrogen utilization.

3.4. Transcriptome Analysis

To reveal the mechanism by which chemical and organic fertilizers regulate growth, the transcripts of the leaves and roots of highland barley were analyzed following OFR0, OFR40, and OFR100 treatments (comparisons between the OFR-treated samples of the leaves and roots tissues are indicated as OFR–L and OFR–R). Analysis of the RNA-seq data revealed 1732 (817 upregulated and 915 downregulated) and 4436 (2618 upregulated and 1818 downregulated) DEGs in the OFR40–L vs. OFR0–L (Figure S1A) and OFR100–L vs. OFR0–L (Figure S2B) groups, respectively. Additionally, 879 (426 upregulated and 453 downregulated) and 2193 (1082 upregulated and 1111 downregulated) DEGs were identified in the OFR40–R vs. OFR0–R (Figure S2C) and OFR100–R vs. OFR0–R (Figure S2D) groups, respectively. The DEGs between the OFR40–L vs. OFR0–L and OFR100–L vs. OFR0–L are shown in Figure S1A. The DEGs between the OFR40–R vs. OFR0–R and OFR100–R vs. OFR0–R are shown in Figure S1B. A pathway enrichment analysis was performed using the KEGG database to gain further insights into the biological functions of the identified DEGs. ‘Photosynthesis’, ‘Fatty acid degradation’, and ‘Glyoxylate and dicarboxylate metabolism’ were the most differentially enriched pathways in OFR40–L vs. OFR0–L (Figure 4A). ‘Carbon fixation in photosynthetic organisms’, ‘Glyoxylate and dicarboxylate metabolism’, and ‘Carbon metabolism’ were the most differentially enriched pathways in OFR100–L vs. OFR0–L (Figure 4B). The expression levels of photosynthesis genes were significantly upregulated in OFR40–L vs. OFR0–L and OFR100–L vs. OFR0–L, indicating that the application of organic fertilizer can increase the expression level of photosynthesis genes in the leaves. The results indicate that the improvement of photosynthetic efficiency converts carbon dioxide into organic matter, thereby increasing yield. ‘Diterpenoid biosynthesis’, ‘Carotenoid biosynthesis’, and ‘Plant hormone signal transduction’ were the most differentially enriched pathways in OFR40–R vs. OFR0–R (Figure 4C). ‘Phenylpropanoid biosynthesis’, ‘Nitrogen metabolism’, and ‘Cysteine and methionine metabolism’ were the most differentially enriched pathways in OFR100–R vs. OFR0–R (Figure 4D). This result indicates that an increase in the concentration of organic fertilizer leads to a shift in differentially expressed genes in roots from secondary metabolism pathways to nitrogen metabolism and amino acid metabolism pathways. The results indicate that the application of organic fertilizer contributes to the absorption and utilization of nitrogen sources by roots, highlighting the potential for organic fertilizers to enhance nutrient efficiency and promote plant growth. Compared to chemical fertilizers, organic fertilizers release nutrients at a slower pace. The metabolic pathways shift from secondary metabolism to primary metabolism in order to meet the basic growth requirements.

3.5. Metabolomic Insights

To investigate significant changes in metabolites of highland barley under different fertilizer treatments, we analyzed the DAMs in the OFR0–L vs. OFR40–L vs. OFR100–L and OFR0–R vs. OFR40–R vs. OFR100–R comparison groups. PCA of the metabolites revealed that the biological replicates were grouped closely, indicating the high reliability of the data. This analysis revealed 735 metabolites in the leaves, including 62 (53 upregulated and 9 downregulated) differentially abundant metabolites in the OFR0–L vs. OFR40–L comparison (Figure S3A) and 66 (17 upregulated and 49 downregulated) in OFR0–L vs. OFR100–L (Figure S3B). With an increase in the concentration of organic fertilizer application, the upregulated metabolic pathways in leaves shift from the ‘Tropane, piperidine, and pyridine alkaloid biosynthesis’ (OFR40–L vs. OFR0–L) to ‘Biosynthesis of amino acids’ pathway (OFR100–L vs. OFR0–L). This shift indicates a metabolic reorientation in response to varying levels of organic fertilizer, potentially enhancing the plant’s ability to synthesize essential amino acids at higher concentrations of organic amendments. A total of 900 metabolites were detected, including 102 (57 upregulated and 45 downregulated) differentially abundant metabolites in the OFR0–R vs. OFR40–R (Figure S3C) and 96 (40 upregulated and 56 downregulated) in the OFR0–R vs. OFR100–R comparisons (Figure S3D). In the roots, with increasing concentrations of organic fertilizer, the upregulated metabolic pathways also shift from the ‘Biosynthesis of secondary metabolites’ pathway to the ‘Phenylalanine, tyrosine, and tryptophan biosynthesis’ and ‘Lysine biosynthesis’ pathways. These findings demonstrate that the application of organic fertilizer in both leaves and roots can shift the metabolic products of barley from the secondary metabolic pathway to the amino acid biosynthesis pathways. This suggests that the increased application of organic fertilizers enhances plant nitrogen uptake and utilization, potentially promoting overall plant growth and health.
The molecular mechanisms regulating growth and carbon and nitrogen assimilation were also studied based on metabolomic analysis. The analysis of the metabolome of leaves showed that with the increasing concentration of organic fertilizer, the metabolic pathways in the leaves shifted from the alkaloid synthesis pathway (OFR0–L vs. OFR40–L) to the amino acid biosynthesis pathway (OFR0–L vs. OFR100–L) (Figure 5A,B). This observation indicated that with reduced chemical fertilizers, non-essential metabolic pathways decreased, while the essential metabolic pathways became more active. The analysis of the metabolome of roots showed that with an increasing concentration of organic fertilizer, the metabolites in the roots shifted from secondary metabolite synthesis and hormone signal transduction pathways (OFR0–R vs. OFR40–R) to amino acid biosynthesis and carbon metabolism pathways, such as the pentose phosphate pathway (OFR0–R vs. OFR100–R) (Figure 5C,D). This observation suggests that with an increasing concentration of organic fertilizer, nutrient absorption and utilization are accelerated in highland barley. The results of our study align with previous reports, indicating that organic fertilizers enhance the nutrient absorption and utilization capabilities of watermelon plants.

3.6. Integrated Analysis of the Transcriptome and Metabolome

To systematically and comprehensively analyze the molecular response in highland barley to different fertilization treatments, we integrated metabolomic and transcriptomic data and mapped the DAMs and DEGs to the same group onto the KEGG pathway. The DAMs and DEGs in the OFR40–L vs. OFR0–L group significantly enriched the pyrimidine metabolism and nicotinate and nicotinamide metabolism pathways (Figure 6A). Meanwhile, the DAMs and DEGs in the OFR100–L vs. OFR0–L group significantly enriched the pathways of cysteine and methionine metabolism and the biosynthesis of amino acids (Figure 6B). The combined analysis of the metabolome and transcriptome revealed that in the OFR0–R vs. OFR40–R comparison group, the DAMs and DEGs were predominantly associated with the flavone and flavonol biosynthesis and the plant hormone signal transduction pathways (Figure 6C). Meanwhile, in the OFR0–R vs. OFR100–R group, the DAMs and DEGs significantly enriched the phenylalanine, tyrosine, and tryptophan biosynthesis and galactose metabolism pathways (Figure 6D). These results indicate that with the increase in organic fertilizer concentration, the DEGs and DAMs shifted from secondary metabolite synthesis to amino acid synthesis and metabolism in leaves and roots.

3.7. Nitrogen Metabolism Gene Expression

Integrative analysis of transcriptomic and metabolomic data indicated that the efficiency of nitrogen uptake and utilization by the roots of highland barley is a significant factor in the regulation of the plant’s growth and development under different fertilization treatments. Therefore, the expression levels of key genes (NR, GS, GOGAT, GOT, and GPT) in nitrogen metabolism in the roots were detected via qRT-PCR under different fertilization conditions. The results indicate that the expression levels of nitrogen metabolism-related genes in the roots exhibit an increasing trend followed by a decreasing trend with the elevation of organic fertilizer concentration, reaching the highest level at OFR40 and showing a decline at OFR100 (Figure 7A–C). However, the expression levels of GOT and GPT genes do not appear to be influenced by different fertilization treatments (Figure 7D,E). The expression levels of NR, GS, and GOGAT genes gradually increase during highland barley growth processes (Figure 7A–C), but there is no significant change pattern in the expression levels of GOT and GPT genes (Figure 7D,E). This suggests that while the nitrogen metabolic pathway is sensitive to the concentration of organic fertilizer, GOT and GPT may be regulated independently of these treatments.

3.8. Nitrogen Metabolism Enzyme Activities

The integrated analysis of the transcriptome and metabolome provided insight into the regulatory mechanisms of nitrogen metabolism in response to different fertilizer treatments. The effects of enzyme activities involved in nitrogen metabolism, including NR, GS, GOGAT, GOT, and GPT, in roots under different fertilizer treatments were observed. The results showed that the activities of enzymes related to nitrogen metabolism, including NR, GS, and GOGAT, significantly increased during the four growth stages (Figure 8A–C), but the activities of GOT and GPT did not change significantly (Figure 8D,E). The enzyme activities of NR, GS, and GOGAT exhibited an initial increase followed by a decrease with the increase in the organic fertilizer level (Figure 8A–C), but there was no significant difference in the activities of GOT and GPT (Figure 8D,E). The results indicate that the application of a low organic fertilizer substitution ratio affects the activities of NR, GS, and GOGAT and impacts the nitrogen absorption and utilization efficiency of plants. However, the application of a high organic fertilizer substitution ratio may reduce nutrient absorption through decreased NR, GS, and GOGAT enzyme activity. This could also explain why the 100% organic fertilizer (OFR100) treatment did not increase the yield of highland barley.

4. Discussion

4.1. Benefits of Organic Fertilizers

The excessive application of fertilizers can result in soil compaction and acidification, which in turn cause an imbalance in soil nutrient levels and the excessive accumulation of certain nutrients. Soil compaction diminishes soil porosity and the availability of oxygen, thereby fostering anaerobic conditions that are conducive to microbial processes. These processes can generate potent greenhouse gases, such as methane (CH4) and nitrous oxide (N2O). Moreover, acidic soils may intensify the emission of nitrous oxide due to microbial nitrification and denitrification activities [32]. Nitrous oxide emissions account for about 6% of total greenhouse gas emissions on a global scale [33]. Therefore, organic fertilizers have been promoted as substitutes for chemical fertilizers. These fertilizers are rich in organic matter and contain nitrogen, phosphorus, potassium, and various micronutrients [34]. The application of organic fertilizers promotes the reproduction of beneficial and active bacteria (Glomeromycota, Mortierellomycota, Humicola, and Bacillus) while inhibiting the reproduction of harmful microorganisms (Ascomycota and Fusarium) and enhances the soil nutrient content, promoting the yield and saponin content [35]. This may reflect the high abundance of beneficial microorganisms in organic fertilizer that can multiply rapidly and become the dominant soil flora, thereby inhibiting the growth of pathogenic fungi. Organic fertilizers have the ability to increase the number of soil aggregates, making the soil structure more porous. This helps to improve the soil’s aeration and permeability, allowing crop roots to grow and breathe more effectively.
Organic fertilizers serve as a food source for soil microorganisms, and their application stimulates the proliferation of these microorganisms in large numbers. These microorganisms can break down the organic matter in the fertilizers, converting it into forms that can be absorbed by crops. Additionally, microbial activity produces growth hormones and enzymes that promote crop growth and development [36]. Thus, the application of organic fertilizers significantly improves crop quality by promoting nutrient balance, coordinating nitrogen supply, and enhancing carbon and nitrogen metabolism [37]. One study showed that in a banana field, the application of organic fertilizers increased soil pH and nutrient availability, as well as yield [38]. In maize, the substitution of organic fertilizer for chemical fertilizers enhanced nitrogen use efficiency and reduced environmental pollution without compromising yield [39]. The use of commercial organic fertilizer promoted plant growth, improved nitrogen and phosphorus use efficiency, and increased both yield and soil quality in wheat fields [40]. Similarly to our study, several studies have reported that organic fertilizers can enhance plant nutrient utilization. The present study found that with the increase in organic fertilizer concentration, the DEGs and DAMs shifted from secondary metabolite synthesis to amino acid synthesis and metabolism.

4.2. Root Plasticity and Nutrient Uptake

Roots are crucial organs in plants that show high developmental plasticity and often help plants adapt to their environment. The distribution and function of roots are crucial for plant survival. In fact, the roots are considered the most limiting parts for plant growth in almost all natural ecosystems. Not surprisingly, the plant root system plays a significant role in yield and overall productivity [41]. Studies have demonstrated that nitrogen limitation promotes taproot growth into deeper soil strata [42], whereas phosphorus deficiency increases the growth of shallow lateral roots [43]. Meanwhile, a study showed that the local availability of nitrates in a nitrate-containing medium stimulated the elongation of lateral roots [44]. This research highlights that applying organic fertilizers increased the length of highland barley roots. Moreover, the DEGs and DAMs in highland barley shifted from a secondary metabolite synthesis pathway to the amino acid synthesis and metabolism pathway in roots. The shift to primary metabolism typically involves increased nitrogen assimilation through the synthesis of amino acids, leading to improved nutrient absorption. The upregulation of amino acid synthesis pathways often results in higher activities of enzymes involved in nitrogen fixation and assimilation. These enzymes facilitate more efficient nitrogen uptake and utilization, significantly enhance nutrient absorption, and promote plant growth. This observation suggests that the root system of highland barley adapts to the nutrient status by elongating the roots. This metabolic shift can enhance root growth by providing essential nutrients, regulating hormonal balance, and promoting beneficial plant microbe interactions. Furthermore, the plant increases its ability to absorb and utilize nutrients. In addition, plant hormone signal transduction (Table S1) was the most differentially enriched pathway, and organic fertilizer-induced plant hormone signal pathways produce hormones such as auxins, gibberellins, and cytokinins, which enhance root development, photosynthesis, stress resistance, and nutrient uptake efficiency. These mechanisms collectively support robust plant growth and higher yields.
Due to soil degradation, global climate change, and inadequate soil inputs, crops in many locations face difficulties in accessing nutrients. Plasticity in root elongation contributes to increased water and nutrient uptake, as it allows for the exploitation of resources over a broader area of the soil profile [45]. Research has shown that fertilizer application affects crop yield and growth conditions, but the specific regulatory mechanisms behind this remain unclear. The current study discovered that using different fertilizers influences nutrient absorption by altering root length. This research provides a theoretical basis for studying the regulation of crop growth conditions using different fertilizers. Previous research has shown that the application of inorganic fertilizers could improve soil microbial community richness, and promote the nutrient uptake and growth of rapeseed [46]. The current study primarily focused on the mechanism by which organic fertilizers enhance plant nutrient uptake to increase yield but did not fully explore the role of soil microbiota in mediating plant growth. Future research should focus on elucidating the mechanisms by which organic fertilizers influence soil microbiota and how these changes can be harnessed to optimize plant growth and yield.

4.3. Implications for Sustainable Agriculture

Excessive use of nitrogen fertilizer has led to a significant decline in crop quality and has contributed to increased nitrate leaching loss, soil acidification, resource wastage, and environmental nitrogen overload. These factors threaten the quality of the environment and human health [47]. A feasible solution to mitigate the harmful impacts of chemical fertilizers and achieve effective crop cultivation is the reduction in nitrogen usage and the replacement of inorganic fertilizers with organic alternatives. This approach can effectively reduce environmental pollution associated with nitrogen fertilizers, promoting efficient agricultural practices [48]. However, the mechanism by which organic fertilizers enhance crop yield is unclear. Findings from this study suggest that applying organic fertilizers promoted the development of highland barley root systems. The presented research findings can provide a theoretical basis for studying the impact of organic fertilizers on plant growth mechanisms. Based on these results, the application of chemical fertilizers combined with organic amendments is recommended as a strategy for improving plant nutrient utilization. In addition, precision agriculture tools can pinpoint low-yielding areas within fields, allowing farmers to apply seeds and nutrients based on zone-specific prescriptions. This approach can reduce wasted inputs and increase profitability by managing inputs at a much finer scale. Controlled-Release Fertilizers are designed to release nutrients slowly over time, ensuring that crops receive the required nutrients throughout their growth cycle. This reduces nutrient loss and improves nutrient use efficiency, leading to higher yields and reduced environmental impact [49].

5. Conclusions

The current study analyzed the impacts of different fertilization schemes on the growth and development of highland barley. This research’s main finding is that the combined application of organic and chemical fertilizers did not only decrease crop productivity but also improved fertilizer utilization efficiency, which is important for providing techniques for the rational and sustainable use of fertilizers. In the future, research should focus on analyzing the mechanism of organic fertilizers, increasing yield to build on these findings and improve the application of mixed fertilization.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15020380/s1: Figure S1: Venn diagram depicting the DEGs of different combinations of 0%, 40%, and 100% organic fertilizers; Figure S2: Effects of different fertilizer treatments on the leaf and root transcriptome of highland barley; Figure S3: Effects of different fertilization treatments on the leaf and root metabolome of highland barley. Table S1. The DEGs list of plant hormone signal transduction in OFR40–R vs. OFR0–R.

Author Contributions

Formal analysis, Q.Y.; investigation, J.Y. and L.H.; project administration, J.Y.; resources, H.W.; software, H.W.; supervision, L.H. and T.H.; writing—original draft, J.Y.; writing—review and editing, T.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Applied Basic Research project of Qinghai province (2022-ZJ-722).

Data Availability Statement

The original contributions presented in the study are publicly available. The data can be found here: [accession number GSE252277].

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Phenotypic characterization of highland barley under three fertilization treatments. (A) Chemical fertilizer at the conventional rate (OFR0), 60% chemical fertilizer + 40% organic fertilizer (OFR40), and 100% organic fertilizer (OFR100) were used in this study; the effects of these treatments on highland barley were analyzed at the seedling stage, jointing stage, heading stage, and flowering stage. (B) The highland barley yield (including the number of grains per spike, 1000-grain weight, and yield per plant) under three fertilization treatments was analyzed. Different letters represent statistically significant differences among OFR0, OFR40, and OFR100 (p < 0.05, two-way ANOVA).
Figure 1. Phenotypic characterization of highland barley under three fertilization treatments. (A) Chemical fertilizer at the conventional rate (OFR0), 60% chemical fertilizer + 40% organic fertilizer (OFR40), and 100% organic fertilizer (OFR100) were used in this study; the effects of these treatments on highland barley were analyzed at the seedling stage, jointing stage, heading stage, and flowering stage. (B) The highland barley yield (including the number of grains per spike, 1000-grain weight, and yield per plant) under three fertilization treatments was analyzed. Different letters represent statistically significant differences among OFR0, OFR40, and OFR100 (p < 0.05, two-way ANOVA).
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Figure 2. Effect of different fertilizer treatments on highland barley nutrient uptake. This study examined the effects of fertilizer treatments, designated as OFR0, OFR40, and OFR100, on the levels of nutrient uptake in highland barley. (A) The nitrogen content in the leaf. (B) The nitrogen content in the root. (C) The phosphorus content in the leaf. (D) The phosphorus content in the root. (E) The potassium content in the leaf. (F) The potassium content in the root. Asterisks indicate statistically significant differences among OFR0, OFR40, and OFR100 (*** p < 0.001, and **** p < 0.0001, one-way ANOVA).
Figure 2. Effect of different fertilizer treatments on highland barley nutrient uptake. This study examined the effects of fertilizer treatments, designated as OFR0, OFR40, and OFR100, on the levels of nutrient uptake in highland barley. (A) The nitrogen content in the leaf. (B) The nitrogen content in the root. (C) The phosphorus content in the leaf. (D) The phosphorus content in the root. (E) The potassium content in the leaf. (F) The potassium content in the root. Asterisks indicate statistically significant differences among OFR0, OFR40, and OFR100 (*** p < 0.001, and **** p < 0.0001, one-way ANOVA).
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Figure 3. Effects of different fertilizer treatments on the C/N ratio of highland barley. This study examined the effects of fertilizer treatments, designated as OFR0, OFR40, and OFR100, on the C/N ratio in highland barley. (A) The C/N ratio in the leaf. (B) The C/N ratio in the root. Asterisks indicate statistically significant differences among OFR0, OFR40, and OFR100 (*** p < 0.001, and **** p < 0.0001, one-way ANOVA).
Figure 3. Effects of different fertilizer treatments on the C/N ratio of highland barley. This study examined the effects of fertilizer treatments, designated as OFR0, OFR40, and OFR100, on the C/N ratio in highland barley. (A) The C/N ratio in the leaf. (B) The C/N ratio in the root. Asterisks indicate statistically significant differences among OFR0, OFR40, and OFR100 (*** p < 0.001, and **** p < 0.0001, one-way ANOVA).
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Figure 4. KEGG pathway analysis shows the top 20 enriched terms associated with the differentially expressed genes (DEGs) of highland barley under different fertilizer treatments. The enriched pathways in the (A) OFR40–L vs. OFR0–L, (B) OFR100–L vs. OFR0–L, (C) OFR40–R vs. OFR0–R, and (D) OFR100–R vs. OFR0–R comparison groups.
Figure 4. KEGG pathway analysis shows the top 20 enriched terms associated with the differentially expressed genes (DEGs) of highland barley under different fertilizer treatments. The enriched pathways in the (A) OFR40–L vs. OFR0–L, (B) OFR100–L vs. OFR0–L, (C) OFR40–R vs. OFR0–R, and (D) OFR100–R vs. OFR0–R comparison groups.
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Figure 5. Bubble diagrams of top 20 KEGG pathways enriched by the differentially accumulated metabolites (DAMs) of highland barley under different fertilizer treatments. The enriched pathways in the (A) OFR40–L vs. OFR0–L, (B) OFR100–L vs. OFR0–L, (C) OFR40–R vs. OFR0–R, and (D) OFR100–R vs. OFR0–R groups. The X-axis represents the p-value, and the Y-axis represents the different pathways. The size of the dots represents the number of differentially accumulated metabolites, and the color of the dot represents the Q-value of enrichment; the deeper the color, the smaller the Q-value.
Figure 5. Bubble diagrams of top 20 KEGG pathways enriched by the differentially accumulated metabolites (DAMs) of highland barley under different fertilizer treatments. The enriched pathways in the (A) OFR40–L vs. OFR0–L, (B) OFR100–L vs. OFR0–L, (C) OFR40–R vs. OFR0–R, and (D) OFR100–R vs. OFR0–R groups. The X-axis represents the p-value, and the Y-axis represents the different pathways. The size of the dots represents the number of differentially accumulated metabolites, and the color of the dot represents the Q-value of enrichment; the deeper the color, the smaller the Q-value.
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Figure 6. KEGG analysis reveals the regulatory mechanism in highland barley under fertilization. The enriched pathways in the (A) OFR40–L vs. OFR0–L, (B) OFR100–L vs. OFR0–L, (C) OFR40–R vs. OFR0–R, and (D) OFR100–R vs. OFR0–R groups. Integrated analysis of the top 20 KEGG pathways enriched by the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs). The X-axis indicates the enrichment score of the DEGs and DAMs. The p-value is presented in a color scale; the sizes of the dots and triangles indicate the number of DEGs and DAMs mapped in each pathway, respectively. The blue arrows indicate amino acid metabolic pathways, and the red arrows indicate the pathways analyzed in detail.
Figure 6. KEGG analysis reveals the regulatory mechanism in highland barley under fertilization. The enriched pathways in the (A) OFR40–L vs. OFR0–L, (B) OFR100–L vs. OFR0–L, (C) OFR40–R vs. OFR0–R, and (D) OFR100–R vs. OFR0–R groups. Integrated analysis of the top 20 KEGG pathways enriched by the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs). The X-axis indicates the enrichment score of the DEGs and DAMs. The p-value is presented in a color scale; the sizes of the dots and triangles indicate the number of DEGs and DAMs mapped in each pathway, respectively. The blue arrows indicate amino acid metabolic pathways, and the red arrows indicate the pathways analyzed in detail.
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Figure 7. Impact of gene expression in nitrogen metabolism pathways of highland barley. This study examines the effects of fertilizer treatments, designated as OFR0, OFR40, and OFR100, on the expression levels of key genes within nitrogen metabolic pathways in highland barley. Measurements were taken at four critical growth stages: the seedling, jointing, heading, and flowering stages. (A) The relative expression level of NR. (B) The relative expression level of GS. (C) The relative expression level of GOGAT. (D) The relative expression level of GOT. (E) The relative expression level of GPT.
Figure 7. Impact of gene expression in nitrogen metabolism pathways of highland barley. This study examines the effects of fertilizer treatments, designated as OFR0, OFR40, and OFR100, on the expression levels of key genes within nitrogen metabolic pathways in highland barley. Measurements were taken at four critical growth stages: the seedling, jointing, heading, and flowering stages. (A) The relative expression level of NR. (B) The relative expression level of GS. (C) The relative expression level of GOGAT. (D) The relative expression level of GOT. (E) The relative expression level of GPT.
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Figure 8. Impact of fertilizer treatments on enzyme activities in nitrogen metabolism pathways of highland barley. This study examines the effects of fertilizer treatments, designated as OFR0, OFR40, and OFR100, on the activity levels of key enzymes within nitrogen metabolic pathways in highland barley. Measurements were taken at four critical growth stages: the seedling, jointing, heading, and flowering stages. The specific enzymes analyzed include the following: (A) the activity of NR; (B) the activity of GS; (C) the activity of GOGAT; (D) the activity of GOT; (E) the activity of GPT.
Figure 8. Impact of fertilizer treatments on enzyme activities in nitrogen metabolism pathways of highland barley. This study examines the effects of fertilizer treatments, designated as OFR0, OFR40, and OFR100, on the activity levels of key enzymes within nitrogen metabolic pathways in highland barley. Measurements were taken at four critical growth stages: the seedling, jointing, heading, and flowering stages. The specific enzymes analyzed include the following: (A) the activity of NR; (B) the activity of GS; (C) the activity of GOGAT; (D) the activity of GOT; (E) the activity of GPT.
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Table 1. Organic and chemical fertilizer content of each treatment.
Table 1. Organic and chemical fertilizer content of each treatment.
TreatmentNutrient Content in Organic Fertilizer (mg·kg−1)Nutrient Content in Chemical Fertilizer (mg·kg−1)
N P2O5K2ON P2O5K2O
OFR0 0 0 0 164.5 185.5 70
OFR40 65.8 12.28 8.94 98.7 173.2 61.06
OFR100 164.5 30.72 22.52 0 154.78 47.48
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Yan, J.; Weng, H.; Hou, L.; Yao, Q.; He, T. Transcriptome and Metabolome Analysis of Organic and Chemical Fertilizer Effects on Highland Barley Growth and Nutrient Utilization. Agronomy 2025, 15, 380. https://doi.org/10.3390/agronomy15020380

AMA Style

Yan J, Weng H, Hou L, Yao Q, He T. Transcriptome and Metabolome Analysis of Organic and Chemical Fertilizer Effects on Highland Barley Growth and Nutrient Utilization. Agronomy. 2025; 15(2):380. https://doi.org/10.3390/agronomy15020380

Chicago/Turabian Style

Yan, Jiahui, Hua Weng, Lu Hou, Qiang Yao, and Tao He. 2025. "Transcriptome and Metabolome Analysis of Organic and Chemical Fertilizer Effects on Highland Barley Growth and Nutrient Utilization" Agronomy 15, no. 2: 380. https://doi.org/10.3390/agronomy15020380

APA Style

Yan, J., Weng, H., Hou, L., Yao, Q., & He, T. (2025). Transcriptome and Metabolome Analysis of Organic and Chemical Fertilizer Effects on Highland Barley Growth and Nutrient Utilization. Agronomy, 15(2), 380. https://doi.org/10.3390/agronomy15020380

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