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Article

Transcriptome Analysis of the Regulatory Mechanism of Exogenous Manganese Sulfate Application on Wheat Grain Yield and Carotenoids

by
Na Yang
1,
Ke Wang
1,
Jiancheng Zhang
1,2,*,
Xiaoyan Jiao
3,
Zhiguo Yang
1,
Jian Wang
1 and
Sha Yang
1
1
The Cotton Research Institute, Shanxi Agricultural University, Yuncheng 044000, China
2
Key Laboratory of Sustainable Dryland Agriculture of Shanxi Province, Taiyuan 030031, China
3
College of Resources and Environment, Shanxi Agricultural University, Taiyuan 030031, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(9), 2190; https://doi.org/10.3390/agronomy15092190
Submission received: 19 August 2025 / Revised: 6 September 2025 / Accepted: 12 September 2025 / Published: 14 September 2025
(This article belongs to the Section Plant-Crop Biology and Biochemistry)
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Abstract

Given the critical role of manganese (Mn) as an essential micronutrient in wheat growth and development and the high efficiency of foliar fertilization in optimizing nutrient uptake and improving crop quality, this study aimed to elucidate the regulatory effects of exogenous manganese sulfate application on wheat grain yield and carotenoid accumulation. Methods: Field experiments were conducted from 2022 to 2024 at the Shuitou Experimental Station of the Cotton Research Institute, Shanxi Agricultural University (35°11′ N, 111°05′ E), using the wheat cultivar ‘Jinmai 110’. Foliar applications of manganese sulfate were administered at concentrations of 0.5 g/kg, 1.0 g/kg, and 1.5 g/kg, with water serving as the control (CTRL). Spraying was conducted on the upper canopy during the flowering and grain-filling stages, applied every 7 days for a total of three times. Samples for transcriptomic analysis were collected within 24 h of the final application. At maturity, yield-related traits and grain carotenoid contents were assessed. Results: Foliar application of 1.0 g/kg MnSO4 significantly enhanced both grain yield and carotenoid content in wheat. Transcriptome sequencing revealed that treatment with 1.0 g/kg manganese sulfate (M2) resulted in 4761 differentially expressed genes (DEGs), including 2933 upregulated and 1828 downregulated genes, relative to CTRL. Gene Ontology (GO) analysis showed that in the M2 vs. CTRL comparison, 819 GO terms were significantly enriched among upregulated DEGs and 630 among downregulated DEGs. Specifically, upregulated genes were associated with 427 biological process terms and 299 cellular component terms, while downregulated genes were linked to 361 biological processes and 211 cellular components. Enriched functions primarily included cellular processes, metabolic processes, catalytic activity, and binding. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed 809 annotations for upregulated DEGs and 330 for downregulated DEGs, mainly related to photosynthesis, carotenoid biosynthesis, phenylpropanoid biosynthesis, and plant hormone signal transduction. In total, 43,395 alternative splicing (AS) events were identified from 17,165 genes, including 445 upregulated and 319 downregulated AS events, primarily enriched in photosynthesis and plant hormone-related pathways. Conclusion: Foliar application of manganese sulfate significantly modulates gene expression in wheat grains, thereby improving both yield and carotenoid accumulation. Key biological processes affected include photosynthesis, plant hormone signal transduction, and the carotenoid biosynthetic pathway. The interactions among these regulatory networks constitute a complex molecular mechanism through which exogenous Mn influences agronomic traits. These findings provide mechanistic insights and practical implications for enhancing wheat productivity and nutritional quality through foliar manganese application.

1. Introduction

With the global population continuously expanding and climate change intensifying, enhancing grain yield and nutritional quality has become a pressing priority for ensuring food security and promoting human health. Yield directly influences agricultural productivity, while carotenoids, as critical components of the plant secondary metabolome, are indispensable for photosystem integrity, redox regulation, and as dietary precursors of vitamin A (e.g., β-carotene) and antioxidant compounds (e.g., lutein and zeaxanthin). Dietary intake of carotenoids contributes to visual health, immune modulation, and the prevention of chronic diseases [1]. Therefore, enhancing the content of carotenoids and their derivatives through methods such as biofortification is an important measure to address human micronutrient deficiencies and protect human health [2]. Biofortification primarily enhances the content of micronutrients in crops by breeding and cultivating. Breeding experts focus on research areas such as varieties’ differences in carotenoid content [3,4], inheritance [5], and functional analysis of key genes [6]. Cultivation methods include management of water [7,8] and fertilizers [9,10], adversity [11,12,13], and exogenous substances [14,15].
Manganese (Mn2+) is an essential trace element for plants, participating in fundamental physiological processes such as photosynthetic electron transport, oxidative phosphorylation, and a broad array of enzyme-mediated reactions [16]. Recently, foliar nutrient application has gained prominence in agriculture due to its efficiency and precision. Exogenous nutrient application via foliar spraying enhances yield and quality by modulating plant hormone dynamics, improving photosynthetic carbon assimilation and optimizing the distribution of assimilates [17,18,19].
In wheat production systems, foliar application of manganese has been reported to significantly improve grain yield and economic return, particularly under no-tillage conditions [20]. In alfalfa, manganese sulfate supplementation has been shown to enhance the yield and nutritional composition [21]. Appropriate concentration of manganese sulfate increases lycopene content in tomato fruits [22]. The regulatory roles of exogenous manganese primarily include: (1) effective foliar uptake that alleviates manganese deficiency and supports normal plant development [23]; (2) its role as a structural component of photosystem II and its key role in photosynthesis [24,25]; (3) activation of the antioxidant defense system, thus enhancing stress tolerance [26]; and (4) regulation of gene expression, thereby improving metabolic efficiency and resilience to abiotic stress [27].
Despite increasing evidence supporting manganese’s role in crop improvement, the underlying molecular mechanisms by which Mn coordinates both yield enhancement and quality improvement remain insufficiently characterized. Exploring a range of manganese concentrations could optimize fertilization strategies tailored for wheat cultivation, while simultaneously advancing sustainable agriculture and soil health [20]. Accordingly, this study applied varying concentrations of foliar manganese sulfate to systematically investigate its effects on wheat yield components, carotenoid biosynthesis, and gene expression profiles. The objective is to provide both theoretical insights and practical strategies for developing green technologies aimed at improving wheat yield and grain quality.

2. Materials and Methods

2.1. Materials

The field experiment was conducted from 2022 to 2024 at the Shuitou Experimental Station of the Cotton Research Institute, Shanxi Agricultural University, located in Xia County (35°11′ N, 111°05′ E), Yuncheng City, Shanxi Province, China. The soil was classified as Calcaric-Fluvic Cambisol [28] with a silty loam texture (28.0% sand, 54.5% silt, and 17.5% clay). The properties of the surface soil (0–20 cm) were as follows: alkali-hydrolyzable nitrogen 0.89 mg/kg, organic matter 12.9 g/kg, available phosphorus 13.1 mg/kg, and available potassium 185.5 mg/kg. The average annual precipitation was 525 mm, the average annual temperature was 13.3 °C, the annual sunshine duration totaled 2293 h, and the frost-free period lasted 212 days.
The experimental plant material was wheat (Triticum aestivum L.), cultivar ‘Jinmai 110’, with seeds supplied by the Wheat Research Group of the Cotton Research Institute, Shanxi Agricultural University. This cultivar is approved by the Shanxi Provincial Variety Committee (approval number: Jinshenmai 20220005). The exogenously applied substance was manganese sulfate monohydrate (MnSO4·H2O:AR, CAS:10034-96-5).

2.2. Experimental Design

Water was used as the control (CK), and three foliar application concentrations of manganese sulfate were tested: 0.5 g/kg (M1), 1.0 g/kg (M2), and 1.5 g/kg (M3). Foliar spraying was applied to wheat spikes during the flowering and grain-filling stages at 7-day intervals for a total of three applications. Each experimental plot covered an area of 15 m2, and the experiment was replicated three times in a randomized complete block design. Upon maturity, grain yield per plot was measured, and carotenoid components—zeaxanthin, lutein, and β-carotene—were quantified. Sowing dates were 18 October 2022 and 23 October 2023, with harvests occurring on 12 June 2023 and 14 June 2024, respectively. Standard field management practices, including pest and disease control, were followed throughout the wheat growth period.

2.3. Measurement Indices and Methods

2.3.1. Wheat Yield

At maturity, five representative sampling points (each 0.667 m2) were harvested from each treatment plot. Wheat is threshed using a thrasher, the model of which is QKT-320A. The moisture content of the grains is detected by a moisture detector (LDS-1G, Shanghai, China). After threshing, grain yield was calculated and adjusted to a standard moisture content of 13%.

2.3.2. Determination of Carotenoid Content in Grains

Carotenoid content in wheat grains was determined according to the method described by Li et al. [29]. The method is Ultra Performance Liquid Chromatography (UPLC).

2.3.3. Transcriptome Sequencing and Data Analysis

Grain samples were collected within 24 h after the third foliar manganese application during the 2023–2024 growing season. Samples were immediately frozen in liquid nitrogen and stored at −80 °C for transcriptome analysis. Total RNA was extracted from the grains of ‘Jinmai 110’ wheat subjected to different manganese sulfate treatments, with three biological replicates per treatment. RNA extraction, cDNA library construction, and sequencing were performed by Beijing Novogene Co., Ltd. (Beijing, China), using the Illumina NovaSeq X Plus platform (Illumina, San Diego, CA, USA). Raw sequencing reads (fastp: version 0.23.1,-g -q 5 -u 50 -n 15 -l 150 --overlap_diff_limit 1--overlap_diff_percent_limit 10) were filtered to obtain high-quality clean reads [30], which were then aligned to the reference genome of Triticum aestivum cv. Chinese Spring (IWGSC RefSeq v2.1) [31] using the HISAT2 software (version 2.2.1) [32]. Only uniquely mapped reads were used for gene expression quantification. Quantitative analysis was performed using the featureCounts tool in the subread software (version 1.5.0-p3) [33]. Differential gene expression analysis was performed using DESeq2 (DESeq2 normalization method, using R package Version: 1.20.0, default parameters, and normalization method is ‘median ratio method’) [34], and gene expression levels were normalized as fragments per kilobase of transcript per million mapped reads (FPKM) [35]. Differentially expressed genes (DEGs) were identified based on a false discovery rate (FDR) ≤ 0.05 and |log2(fold change)| ≥ 1. Differential alternative splicing (AS) events were analyzed using rMATS v4.1.0 [36], covering five AS types: skipped exon (SE), alternative 3′ splice site (A3SS), alternative 5′ splice site (A5SS), retained intron (RI), and mutually exclusive exons (MXE). rMATS calculates exon inclusion levels (ψ) for each sample, and differential AS events were defined by FDR < 0.05 and |Δψ| > 0.1. Functional annotation and enrichment of differentially expressed genes (DEGs) between treatments were conducted using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. GO terms and KEGG pathways with p ≤ 0.05 were considered significantly enriched. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler (4.8.1), in which gene length bias was corrected. GO terms with corrected p-value less than 0.05 were considered significantly enriched by differential expressed genes. By default, the Benjamini–Hochberg (BH) method was used to correct the p-values, which was achieved through the p.adjust function.
  • padjust = p.adjust (p,method = “BH”)
  • HISAT2 version 2.2.1: hisat2 -x <hisat2 index> -p 4 --dta -t --phred33 -1 sample_1.clean.fq.gz -2 sample_2.clean.fq.gz --un-conc-gz sample.unmap.fq.gz 2> sample_align.log | samtools sort -O BAM --threads 4 -o sample.bam -. These were used as default parameters.

2.4. Data Processing

Data were organized using WPS Office Excel. Analysis of variance (ANOVA) was performed using DPS 9.0 software. The method used for the analysis of variance (ANOVA) was Tukey’s test. Graphs were generated using Origin 2024.

3. Results

3.1. Effects of Foliar Application of Different Concentrations of Manganese Sulfate on Wheat Grain Yield

The influence of foliar application of manganese sulfate at various concentrations during the flowering and grain-filling stages on wheat grain yield is presented in Figure 1. In the 2022–2023 growing season, compared with the control (CTRL), grain yield was significantly reduced under the 0.5 g/kg manganese sulfate treatment (M1), whereas no significant difference was observed for the 1.5% treatment (M3) (p > 0.05). In contrast, the 1.0 g/kg manganese sulfate treatment (M2) significantly increased grain yield by 5.52% compared to the control (p < 0.05). In 2023–2024, grain yield under the M2 treatment increased by 1.85% relative to the control, while a decrease was observed in M3. A significant 3.81% increase was observed in the two-year average yield (p < 0.05). These findings suggest that an appropriate concentration of manganese sulfate (1.0 g/kg) promotes wheat grain yield, whereas higher concentrations do not confer additional benefits.

3.2. Effects of Foliar Application of Different Concentrations of Manganese Sulfate on Carotenoid Content in Wheat Grains

As shown in Figure 2, the application of manganese sulfate at different concentrations differentially affected the levels of zeaxanthin, lutein, and β-carotene in wheat grains. In the 2022–2023 season, the 1.0 g/kg manganese sulfate treatment (M2) had no significant effect on zeaxanthin content compared to the control but significantly increased lutein and β-carotene contents by 22.83% and 7.41%, respectively. M1 had no significant effect on lutein and β-carotene contents compared to the control; M3 had decreased lutein content compared to the control but had no significant effect on β-carotene content. In contrast, zeaxanthin content decreased under both the M1 and M3 treatments. In 2023–2024, M2 again showed positive effects, increasing lutein and β-carotene contents by 4.60% and 16.67%, respectively, compared with the control. M1 and M3 decreased the content of lutein significantly and had no significant effect on β-carotene content, but it increased the zeaxanthin content significantly.
Across both experimental years, zeaxanthin levels exhibited a slight upward trend with increasing manganese sulfate concentrations. M2 was the most effective treatment in increasing lutein content. Collectively, these results indicate that foliar application of manganese sulfate at an optimal concentration (1.0 g/kg) promotes carotenoid biosynthesis in wheat grains, whereas suboptimal (too low or too high) concentrations may lead to neutral or negative effects.

3.3. Transcriptome Sequencing Analysis of Wheat Grains Under Foliar Application of Exogenous Manganese Sulfate

3.3.1. Quality Assessment of Sequencing Data

Totals of 41,795,375; 42,981,606; 43,631,029; and 44,238,796 raw reads were generated for the CTRL, M1, M2, and M3 treatments, respectively (Table S1). After removing adapter sequences, reads containing ≥10% ambiguous bases, and low-quality reads, the number of clean reads retained were 40,285,289 (CTRL), 41,437,978 (M1), 42,152,064 (M2), and 42,507,406 (M3), corresponding to 97.94% and 98.08% of the raw reads. The combined dataset for the 12 samples yielded 142.10 Gb of clean sequencing data. The proportion of bases with Q20 quality scores exceeded 96.1% across all samples, while Q30 quality scores were greater than 90.05%, indicating high sequencing accuracy and data quality suitable for downstream transcriptomic analyses. The R2 of all samples (Figure 3) was greater than 0.87, indicating good biological reproducibility of the samples.

3.3.2. Statistical Analysis of DEGs

Figure 4, Figure 5 and Figure 6 present volcano plots illustrating the differential expression of genes across treatments. As evident in the comparisons of M1 vs. CTRL, M2 vs. CTRL, and M3 vs. CTRL, the number and direction of DEGs varied with manganese sulfate concentration. Notably, the number of upregulated genes exceeded that of downregulated genes in both M1 and M2 comparisons. According to the DEG screening criteria (FDR ≤ 0.05 and |log2FC| ≥ 1), the following numbers of DEGs were identified: in M1 vs. CTRL, a total of 3144 DEGs were identified (1742 upregulated, 1402 downregulated); in M2 vs. CTRL, a total of 4761 DEGs were identified (2933 upregulated, 1828 downregulated); in M3 vs. CTRL, a total of 5498 DEGs were identified (2474 upregulated, 3024 downregulated). These results indicate that the number of differentially expressed genes varies when different concentrations of manganese sulfate are sprayed. Compared to the control, M2 treatment yielded the highest number of upregulated genes, while M3 showed the largest number of downregulated genes and all differentially expressed genes.

3.3.3. GO Classification and Enrichment Analysis of Differentially Expressed Genes

To further elucidate the biological mechanisms underlying the observed differences in wheat grain yield and carotenoid content following foliar application of manganese sulfate—and in conjunction with the previous findings regarding the effects of different manganese concentrations on yield and carotenoid accumulation—GO annotation and enrichment analyses were performed for the DEGs identified in the M2 vs. CTRL comparison. GO annotation was carried out across three major categories: biological process (BP), molecular function (MF), and cellular component (CC), followed by functional enrichment analysis. Figure 7 and Figure 8 illustrate the GO enrichment results for upregulated and downregulated DEGs, respectively, in the M2 vs. CTRL comparison. A total of 819 significantly enriched GO terms were identified among the upregulated DEGs, distributed across 427 BP terms, 93 MF terms, and 299 CC terms. For the downregulated DEGs, 630 enriched GO terms were detected, including 361 in BP, 58 in MF, and 211 in CC. Within the BP category, upregulated DEGs were primarily enriched in DNA replication, fatty acid metabolism and biosynthesis, intracellular signal transduction, and phosphotransferase-mediated signaling—pathways associated with grain development, stress response, and metabolic activity. In contrast, downregulated DEGs were enriched in pathways related to abiotic stress responses, dicarboxylate transport, and 1,3-β-D-glucan biosynthesis and metabolism, suggesting that manganese sulfate treatment may modulate both stress adaptation and carbohydrate metabolism. In the CC category, upregulated DEGs were associated with DNA–protein complexes, membrane-anchored components, and the apoplast. Downregulated DEGs were enriched in components such as the myosin complex, plasma membrane, cytoskeleton, and transferase complexes. These results imply that foliar manganese sulfate alters gene expression in distinct subcellular compartments, with upregulated genes often linked to nuclear and structural components, and downregulated genes associated with membrane and cytoskeletal structures.
For MF, upregulated DEGs showed significant enrichment in glucosidase activity, hydrolase activity, transferase activity, and calcium ion binding. In contrast, downregulated DEGs were mainly involved in ligase activity, carbon–nitrogen bond formation, transporter activity, hydrolase activity, and kinase activity. These patterns suggest that 1.0 g/kg manganese sulfate treatment promotes the activity of multiple enzymes.

3.3.4. The KEGG Pathway Analysis of Significant Differentially Expressed Genes

To further understand the functional implications of the identified DEGs, KEGG pathway enrichment analysis was conducted for both upregulated and downregulated DEGs in the M2 vs. CTRL comparison. Among the 2933 upregulated DEGs, 809 (27.58%) were successfully annotated in the KEGG database. These genes were associated with 111 metabolic pathways, of which 11 were significantly enriched (Figure 9). Key enriched pathways included fatty acid elongation, ATP-dependent chromatin remodeling, DNA replication, motor proteins, photosynthesis–antenna proteins, nucleoside sugar biosynthesis, galactose metabolism, ascorbate and aldarate metabolism, fatty acid biosynthesis, phagosome formation, and lipoic acid metabolism. Additional enrichment was observed in processes related to calcium ion binding, transferase activity, the apoplast, the endoplasmic reticulum, and cellular signal transduction—many of which are directly involved in carotenoid biosynthesis and related metabolic regulation.
Of the 1828 downregulated DEGs, 330 (18.05%) were annotated in the KEGG database. These genes were mapped to 95 metabolic pathways, with significant enrichment in seven pathways, including galactose metabolism, sulfur metabolism, nucleotide metabolism, cysteine and methionine metabolism, tyrosine metabolism, and purine metabolism. Other pathways impacted included the pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, exopolysaccharide biosynthesis, pentose and glucuronate interconversions, flavonoid biosynthesis, and pyruvate metabolism (Figure 10).
Notably, the galactose metabolism pathway was significantly enriched in both upregulated (24 genes) and downregulated (16 genes) DEGs, indicating a complex, bidirectional regulation of this pathway under 1.0 g/kg manganese sulfate treatment. These findings demonstrate that exogenous manganese sulfate significantly modulates key pathways involved in lipid, sugar, and secondary metabolite metabolism in wheat grains.
The GSEA enrichment analysis (Figure 11 and Figure 12) based on the KEGG gene set shows that the M2 treatment ES in the carotenoid biosynthesis pathway and the photosynthetic antenna protein pathway is greater than 0 (ES > 0), and the barcodes are displayed in red, indicating that the gene sets of both pathways are significantly upregulated in M2 treatment. However, ES < 0 and the blue barcode in the control indicate that the control treatment significantly downregulated the gene sets of the two pathways.

3.3.5. Alternative Splicing Analysis

Analysis of Alternative Splicing Events
Using the same RNA-seq dataset, a comprehensive analysis of AS events was conducted for the M2 vs. CTRL comparison. The analysis revealed a total of 43,395 AS events across 23,141 genes (Table 1). Among these, the most prevalent AS type was SE, accounting for 30.14% of total AS events. This was followed by A3SS (26.41%), RI (25.82%), MXE (16.13%), and A5SS (1.50%).
To explore changes in splicing regulation under manganese sulfate treatment, differential AS events were analyzed between M2 and CTRL. Totals of 445 upregulated and 319 downregulated AS events were identified (Figure 13), suggesting that manganese treatment affects post-transcriptional regulatory mechanisms in addition to transcriptional control.
The GO and KEGG Functional Enrichment Analysis of Differentially Spliced Genes
To investigate the functional consequences of AS events regulated by manganese sulfate treatment, GO and KEGG enrichment analyses were performed for differentially spliced genes in the M2 vs. CTRL comparison. The GO enrichment revealed a total of 308 significantly enriched terms. Within the MF category, terms related to transferase activity and phosphoglycerate kinase activity were prominent, indicating that AS events affect functions essential for energy conversion and metabolic processing. Enriched CC terms included RNA polymerase III complexes, the actin cytoskeleton, and DNA-directed RNA polymerase complexes, which are crucial for cellular structure and transcriptional regulation. In the BP category, AS-regulated genes were enriched in terms related to organelle organization, suggesting enhanced development and maintenance of subcellular compartments in wheat grains under manganese treatment.
In the KEGG analysis, 68 enriched pathways were identified, including glycosylphosphatidylinositol (GPI)–anchor biosynthesis, pyrimidine metabolism, the biosynthesis of phenylalanine, tyrosine, and tryptophan, and the pentose phosphate pathway (Figure 14 and Figure 15). Pathways involving aromatic amino acids are central to both protein biosynthesis and the production of secondary metabolites, while the pentose phosphate pathway is integral to carbohydrate metabolism and energy provision. These results suggest that manganese sulfate application not only affects gene expression but also post-transcriptional splicing, potentially influencing key metabolic and physiological outcomes in wheat grains.

4. Discussion

In this study, the wheat cultivar ‘Jinmai 110’ was used to investigate the effects of foliar application of different concentrations of manganese sulfate on grain yield and carotenoid accumulation. By integrating transcriptome analysis of DEGs and AS events between the 1.0 g/kg manganese sulfate treatment and the control (CTRL), this study aimed to elucidate the molecular mechanisms by which foliar-applied Mn influences yield and carotenoid content, thereby providing a theoretical foundation for its rational use in wheat production.
As an essential micronutrient, manganese plays a critical role in wheat growth and development. In the present study, foliar application of an optimal concentration of manganese sulfate significantly enhanced grain yield, likely through the stimulation of key physiological processes such as photosynthesis, assimilate translocation, and grain filling. Previous studies have reported similar outcomes; for example, treatment with 1.0–1.5 mg/L MnSO4 significantly increased the chlorophyll content in wheat seedlings, thereby promoting vegetative growth [37]. Zhang et al. [38] also demonstrated that foliar Mn application during grain filling significantly improved final yield. Zeng et al. [39] reported yield increases of 29% following 0.2% MnSO4 application, further supporting its positive agronomic effect. Moreover, other studies suggest that manganese contributes to enhanced stress tolerance in wheat [40]. Consistent with these findings, the current study showed that both 1.0 g/kg and 1.5 g/kg manganese sulfate treatments significantly increased yield. However, the absence of a significant difference between these two treatments suggests a concentration threshold beyond which additional manganese may not enhance yield further, indicating a possible saturation effect [41] or toxicity [42].
Carotenoids serve as key nutritional quality indicators in wheat, contributing not only to human health but also to plant stress tolerance via their antioxidant properties. While several studies have investigated the effects of other nutrients such as silicon [40], zinc [43], iron–zinc complexes [44], and abscisic acid [45] on carotenoid accumulation, reports on manganese sulfate remain limited. Nevertheless, research has shown that exogenous Mn can enhance chlorophyll and carotenoid levels in barnyard grass under saline-alkali stress [41] and increase carotenoid content in sweet potato [46]. In line with these findings, this study revealed that foliar application of 1.0 g/kg manganese sulfate significantly increased lutein and β-carotene concentrations in wheat grains.
Transcriptome analysis of the M2 vs. CTRL comparison revealed that manganese sulfate treatment had a broad impact on gene expression in wheat grains. GO and KEGG enrichment analyses indicated that both upregulated and downregulated genes were enriched in metabolic pathways associated with yield formation and grain quality. For instance, upregulated DEGs were significantly enriched in the photosynthesis antenna proteins pathway, which plays a pivotal role in light energy capture and directly contributes to yield enhancement. Carotenoids are essential components in photosynthetic systems and play a critical role in the function of antenna proteins [47]. These photosynthetically active antenna proteins capture light energy and efficiently transfer it to the reaction center [48]. In this study, the enrichment of genes associated with photosynthetically active antenna proteins suggests not only that manganese application can enhance photosynthetic capacity in wheat, but also that it may promote increased carotenoid biosynthesis. The activity of geranylgeranylpyrophosphate synthase (GGPS) depends on Mn2+ [49], thereby influencing the key regulatory factor -Geranylgeranyl pyrophosphate (GGPP) [50]. Additionally, pathways such as galactose metabolism and ascorbate and aldarate metabolism—also enriched among upregulated genes—are involved in the synthesis and distribution of carbohydrates and antioxidants, potentially improving grain nutritional quality. In contrast, downregulated DEGs were enriched in energy-related pathways such as the pentose phosphate pathway and glyoxylate and dicarboxylate metabolism, indicating a possible shift in energy allocation under manganese treatment. Of particular interest was the dual enrichment of the galactose metabolism pathway among both upregulated and downregulated DEGs, suggesting a nuanced regulatory mechanism in carbohydrate partitioning. Previous research has linked modulation of this pathway to changes in acetyl-CoA availability, which in turn can influence carotenoid biosynthesis, aligning with the observed increase in β-carotene content [51].
Transcriptome sequencing also revealed widespread alterations in alternative splicing patterns. As a key post-transcriptional regulatory mechanism, alternative splicing can generate transcript isoform diversity, fine-tune metabolic regulation [52] and the function of organelle organization. Carotenoids are mainly synthesized in the plastids of plant cells [53]. The endoplasmic reticulum is mainly involved in protein synthesis, processing and the metabolism of lipids (including carotenoids). For instance, the smooth endoplasmic reticulum can synthesize carotenoid precursor substances [54].
In this study, manganese sulfate induced numerous AS events affecting genes involved in diverse biological processes and metabolic pathways, underscoring the complexity of its regulatory role in wheat grain development.
In summary, transcriptome and AS event analyses demonstrated that manganese sulfate application induces extensive molecular changes in wheat grains, impacting gene expression and regulatory networks associated with yield and nutritional quality. These findings provide valuable insights into the functional role of micronutrient regulation in crop performance.
Although the current results confirm the beneficial effects of foliar MnSO4 on wheat grain yield and carotenoid content and offer initial insights into its molecular mechanisms, the field efficacy of manganese sulfate may be influenced by environmental factors such as soil properties, climatic conditions, and varietal differences. Furthermore, current transcriptomic analyses related to carotenoid biosynthesis in wheat are limited by the absence of specialized carotenoid-targeted datasets [55]. Future research should therefore investigate the application of manganese sulfate under diverse agroecological conditions and integrate multi-omics approaches, such as proteomics and metabolomics, to comprehensively elucidate the regulatory mechanisms of carotenoid accumulation and yield enhancement in wheat.

5. Conclusions

This study investigated the effects of foliar application of varying concentrations of manganese sulfate on wheat grain yield, carotenoid accumulation, and transcriptomic responses. The results demonstrated that treatment with 0.10% manganese sulfate significantly enhanced both grain yield and carotenoid content. Transcriptome analysis revealed extensive changes in gene expression and alternative splicing events, with DEGs associated with key metabolic pathways including photosynthesis, carbohydrate metabolism, and energy regulation. Alternative splicing further contributed to the modulation of metabolic processes at the post-transcriptional level. Together, these findings highlight the potential of foliar manganese sulfate application to simultaneously improve both agronomic and nutritional traits in wheat. This study offers a theoretical framework for the judicious and effective use of manganese sulfate in wheat production systems.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy15092190/s1.

Author Contributions

Conceptualization, J.Z. and N.Y.; methodology, J.Z. and K.W.; software, S.Y.; validation, N.Y., J.Z. and Z.Y.; formal analysis, S.Y.; investigation, Z.Y. and K.W.; resources, J.W.; data curation, J.W. and K.W.; writing—original draft preparation, N.Y.; writing—review and editing, K.W.; supervision, X.J. and J.Z.; project administration, X.J.; funding acquisition, X.J. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Key Research Project of Shanxi Province, grant number 2022ZDYF108; the Project of Shanxi Province Key Lab Construction, grant number Z135050009017-1-15; the National Key Research and Development Program of China, grant number 2021YFD1901102; and the Project of Yuncheng City Basic Research (YCKJ-2023037).

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Acknowledgments

We gratefully thank the anonymous reviewers and editors for their helpful comments that greatly improved this manuscript. During the preparation of this manuscript/study, we used DPS9.0 for the purposes of analysis of variance (ANOVA) and Origin 2024 for the purposes of generating graphs. We have reviewed and edited the output and take full responsibility for the content of this publication. In the preparation of this manuscript, we utilized Deepseek (Deepseek R1) for the purpose of polishing the language of this manuscript. We have carefully reviewed and verified all content generated by the GenAI tool to ensure its accuracy, consistency, and compliance with academic integrity. We are responsible for the final content of this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
DEGsDifferentially expressed genes
FDRFalse discovery rate
SESkipped exon
A3SSAlternative 3′ splice site
A5SSAlternative 5′ splice site
RIRetained intron
MXEMutually exclusive exons
GOGene Ontology
KEGGKyoto Encyclopedia of Genes and Genomes
FPKMFragments per kilobase of transcript per million mapped reads
ESEnrichment Score
GSEAGene Set Enrichment Analysis

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Figure 1. Effects of different treatments on wheat grain yield (kg/ha). Different lowercase letters indicate significant differences between treatments (p < 0.05). The error line shows the standard deviation of these repeated measurements.
Figure 1. Effects of different treatments on wheat grain yield (kg/ha). Different lowercase letters indicate significant differences between treatments (p < 0.05). The error line shows the standard deviation of these repeated measurements.
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Figure 2. Carotenoid content in wheat grains under different treatments. Different lowercase letters indicate significant differences between treatments (p < 0.05). The error line shows the standard deviation of these repeated measurements.
Figure 2. Carotenoid content in wheat grains under different treatments. Different lowercase letters indicate significant differences between treatments (p < 0.05). The error line shows the standard deviation of these repeated measurements.
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Figure 3. Correlation heat map.
Figure 3. Correlation heat map.
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Figure 4. Summary of the number of upregulated and downregulated DEGs in M1 vs. CTRL.
Figure 4. Summary of the number of upregulated and downregulated DEGs in M1 vs. CTRL.
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Figure 5. Summary of the number of upregulated and downregulated DEGs in M2 vs. CTRL.
Figure 5. Summary of the number of upregulated and downregulated DEGs in M2 vs. CTRL.
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Figure 6. Summary of the number of upregulated and downregulated DEGs in M3 vs. CTRL.
Figure 6. Summary of the number of upregulated and downregulated DEGs in M3 vs. CTRL.
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Figure 7. GO enrichment analysis of up-regulated DEGs in M2 vs. CTRL.
Figure 7. GO enrichment analysis of up-regulated DEGs in M2 vs. CTRL.
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Figure 8. GO enrichment analysis of down-regulated DEGs in M2 vs. CTRL.
Figure 8. GO enrichment analysis of down-regulated DEGs in M2 vs. CTRL.
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Figure 9. KEGG enrichment analysis of up-regulated DEGs in M2 vs. CTRL.
Figure 9. KEGG enrichment analysis of up-regulated DEGs in M2 vs. CTRL.
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Figure 10. KEGG enrichment analysis of down-regulated DEGs in M2 vs. CTRL.
Figure 10. KEGG enrichment analysis of down-regulated DEGs in M2 vs. CTRL.
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Figure 11. GSEA enrichment analysis score chart based on KEGG gene set (Carotenoid_biosynthesis).
Figure 11. GSEA enrichment analysis score chart based on KEGG gene set (Carotenoid_biosynthesis).
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Figure 12. GSEA enrichment analysis score chart based on KEGG gene set (Photosyhthesis_antenna_proteins).
Figure 12. GSEA enrichment analysis score chart based on KEGG gene set (Photosyhthesis_antenna_proteins).
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Figure 13. Number of differential alternative splicing events in M2 vs. CTRL.
Figure 13. Number of differential alternative splicing events in M2 vs. CTRL.
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Figure 14. GO enrichment analysis of alternative splicing events.
Figure 14. GO enrichment analysis of alternative splicing events.
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Figure 15. KEGG enrichment analysis of alternative splicing events.
Figure 15. KEGG enrichment analysis of alternative splicing events.
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Table 1. Summary of alternative splicing events in M2 vs. CTRL.
Table 1. Summary of alternative splicing events in M2 vs. CTRL.
AS EventGene NumberAS Number
SE758713,080
MXE369652
A5SS38986999
A3SS616311,459
RI512411,204
Total23,14143,394
Note: skipped exon (SE), alternative 3′ splice site (A3SS), alternative 5′ splice site (A5SS), retained intron (RI), and mutually exclusive exons (MXE).
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MDPI and ACS Style

Yang, N.; Wang, K.; Zhang, J.; Jiao, X.; Yang, Z.; Wang, J.; Yang, S. Transcriptome Analysis of the Regulatory Mechanism of Exogenous Manganese Sulfate Application on Wheat Grain Yield and Carotenoids. Agronomy 2025, 15, 2190. https://doi.org/10.3390/agronomy15092190

AMA Style

Yang N, Wang K, Zhang J, Jiao X, Yang Z, Wang J, Yang S. Transcriptome Analysis of the Regulatory Mechanism of Exogenous Manganese Sulfate Application on Wheat Grain Yield and Carotenoids. Agronomy. 2025; 15(9):2190. https://doi.org/10.3390/agronomy15092190

Chicago/Turabian Style

Yang, Na, Ke Wang, Jiancheng Zhang, Xiaoyan Jiao, Zhiguo Yang, Jian Wang, and Sha Yang. 2025. "Transcriptome Analysis of the Regulatory Mechanism of Exogenous Manganese Sulfate Application on Wheat Grain Yield and Carotenoids" Agronomy 15, no. 9: 2190. https://doi.org/10.3390/agronomy15092190

APA Style

Yang, N., Wang, K., Zhang, J., Jiao, X., Yang, Z., Wang, J., & Yang, S. (2025). Transcriptome Analysis of the Regulatory Mechanism of Exogenous Manganese Sulfate Application on Wheat Grain Yield and Carotenoids. Agronomy, 15(9), 2190. https://doi.org/10.3390/agronomy15092190

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