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Article

Effects of Ammonium on Assimilate Translocation and Storage Root Growth in Sushu16 in Root-Swelling Stage

by
Wenjing Yao
1,2,3,
Rui Zhou
1,
Qin Tan
1,
Chun Zhuang
2,
Wenqi Shao
2,
Chuan Chen
2 and
Chuanzhe Li
2,*
1
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
2
Huaiyin Institute of Agricultural Sciences of Xuhuai Area, Jiangsu Academy of Agricultural Science, Huaian 223001, China
3
State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China
*
Author to whom correspondence should be addressed.
Agronomy 2025, 15(6), 1272; https://doi.org/10.3390/agronomy15061272
Submission received: 28 April 2025 / Revised: 19 May 2025 / Accepted: 20 May 2025 / Published: 22 May 2025
(This article belongs to the Section Soil and Plant Nutrition)
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Abstract

Ammonium greatly influences nutrient partitioning and root architecture, particularly in the tuberous crops where assimilate translocation is critical for yield formation. However, relatively few studies have systematically delved into the physiological and molecular mechanisms of ammonium on assimilate translocation and root growth in sweetpotato (Ipomoea batatas Lam.). In this study, we investigated the morphological, physiological, and molecular effects of different concentrations of ammonium (0, 0.5, 1.0, 3.0, 5.0 mM) on the growth of the Sushu16 variety in the root-swelling stage. The plant weight and leaf area index of Sushu16 seedlings exhibited a progressive increase with elevated ammonium levels. However, the weight, volume, and number of storage roots (SRs) displayed a trend of a rapid rise and substantial decline, peaking at 1 mM ammonium. Similarly, the chlorophyll content, photosynthetic rate, and stomatal conductance were significantly increased with 1 mM ammonium treatment. Further, the contents of CK, ABA, and IAA increased first and then decreased, reaching a maximum at 1 mM ammonium. Notably, the “down then up” trend of sucrose content in leaves and stems contrasted with the fall–rise pattern of starch content in SRs at 1 mM ammonium. Furthermore, we screened 34 significant DEGs involved in photosynthesis, starch biosynthetic processes, and hormone signal pathway in SRs by RNA-Seq. All the results indicated that 1 mM ammonium had a promotive effect on source–sink conversion and SR production in Sushu16, which highlights potential targets for breeding or agronomic strategies to optimize yield formation in sweetpotato.

1. Introduction

Sweetpotato (Ipomoea batatas Lam.) is one of the most important food crops in the world. According to the report of the Food and Agriculture Organization (FAO), its global production exceeded 140 million tons in 2019. Asia is the largest producing region of sweetpotato, accounting for 75% of global production [1]. Particularly, China is the biggest cultivator country of sweetpotato. In 2022, its production amounted to 46.8288 million tons in China, contributing to 54.19% of the world’s output [2]. Sweetpotato displays advantages of high productivity, low inputs, and strong tolerance to unfavorable climatic and cultivation conditions. It has versatile uses and economic values, such as human consumption, various product (starch, noodles, natural colorants, alcohol, etc.) processing, and animal feed [3]. The crop is primarily grown for its edible storage roots (SRs), which are rich in vitamins (vitamin C, B complex, and E), minerals (potassium, calcium, and iron), and carbohydrates [4]. Starch is one of the primary carbohydrate sources, which occupies 50–80% of the dry matter of SRs in sweetpotato [5]. The massive conversion and filling processes of starch are fundamental to the formation and yield potential of SRs in sweetpotato [6]. The root-swelling stage is a critical period of photosynthate translocation from the source to sink in sweetpotato [7]. A large sink capacity and rapid assimilate translocation from the source to the sink in the root-swelling stage contribute to high yield in sweetpotato [8].
Nitrogen (N), being “the motor of plant growth”, is an essential mineral macroelement, playing critical roles in almost all metabolic activities in plants [9]. The absorption of N is highest among the essential elements in crop plants, which is one of the most limiting factors in crop yield [10]. N deficiency is not conducive to the formation and development of root tubers, leading to reduced yield formation in sweetpotato [11]. However, excessive N promotes aboveground overgrowth and inhibits root swelling, reducing SR production in sweetpotato [12,13,14]. In addition, as an important component of chlorophyll, N indirectly affects photosynthetic characteristics in plants [15]. There is a significant positive correlation between dry biomass and photosynthetic rate (Pn) in sweetpotato [5]. Therefore, optimizing the N fertilizer is of particular importance in enhancing the yield of sweetpotato.
Unveiling the specific mechanisms of action in N absorption, distribution, and utilization is a prerequisite for effective N fertilizer in agriculture production. Plant roots can take up and assimilate N in inorganic (nitrate and ammonium) and organic (e.g., urea, amino acids, peptides) forms [16]. The main forms of N sources are nitrate, ammonium, and urea in agricultural fertilization, which exhibit significant differences in root uptake, translocation rate, and N metabolism, ultimately altering plant growth and development [17]. The uptake rate of nitrate or ammonium is significantly higher than that of urea as the sole N form. Ammonium application enhances the translocation rate of N and cytokinins (CKs), compared to urea [18]. Ammonium is the preferred N source over nitrate, as it displays the advantages of permanent availability [10]. In addition, ammonium assimilation requires less energy as it is already in reduced form, potentially redirecting metabolic resources toward carbon fixation [19].
The moderate external application of ammonium promotes plant growth [17]. Notably, the ammonium supply modifies the root system architecture by inhibiting primary root growth and stimulating lateral root branching or swelling root hairs [20,21]. With the same dosage, ammonium treatment induced a greater increase in SR production by increasing the number of SRs in sweetpotato, compared to amide N [16]. However, exclusive ammonium results in considerable developmental changes, such as root growth inhibition, accelerated flowering, tiller number decrease, seed yield decline, etc. [18,22]. Especially as a sole N source, elevated ammonium supplies can rapidly induce various metabolic and hormonal imbalances that ultimately inhibit root growth in many plant species [21,23]. For example, prolonged exclusive ammonium causes a dramatically decreased auxin response in primary roots, leading to root growth inhibition in Arabidopsis [23].
Several studies have confirmed that moderate ammonium application contributes to yield formation in different sweetpotato cultivars [11,13,16]. However, relatively few studies have systematically delved into the physiological and molecular mechanisms of ammonium application on assimilate translocation and root growth in sweetpotato. Sushu16 is one of the representative excellent fresh-eating varieties cultivated by Jiangsu Academy of Agricultural Sciences, which displays advantages of mechanical and physical properties such as the moisture content and compressive strength limit [14]. This study takes SuShu16 transplants in the root-swelling stage as the experimental subjects, to characterize the effects of different concentrations of ammonium on the growth of sweetpotato at morphological, physiological, and molecular levels. Under the contents set up (i.e., 0, 0.5, 1.0, 3.0, 5.0 mM), Sushu16 seedlings achieved optimal morphological traits and physiological states at a 1 mM ammonium concentration, such as significantly increased SR biomass, SR starch content, chlorophyll content, Pn, and hormone contents. In addition, a large number of differentially expressed genes (DEGs) were profiled in SRs with ammonium treatments by RNA-Seq, which were annotated to participate in photosynthesis (76 genes), carbon metabolism (536 genes), starch and sucrose metabolism (221 genes), carbon fixation in photosynthetic organisms (127 genes), and hormone signal transduction (217 genes). Of particular importance was the identification of 34 significant DEGs involved in photosynthesis (10 genes), starch biosynthetic processes (19 genes), and the hormone signal pathway (five genes), leading to enhanced Pn, increased starch accumulation, and improved hormone contents in SRs. All the results indicate that a dose of 1 mM ammonium can effectively improve photosynthesis, promote sucrose–starch conversion, and activate the expression of key genes related to starch biosynthesis in Sushu16, which not only lays a foundation for ammonium fertilizer optimization in sweetpotato, but also provides new insights into the molecular physiology of ammonium on root growth and yield formation in tuberous crops.

2. Materials and Methods

2.1. Plant Materials

Clonal propagation cuttings of Sushu16 cultivar were pot-cultured under greenhouse conditions with 60–70% humidity, 16:8 h light/dark cycles, and an ambient temperature of 24 ± 2 °C in the experimental field of Jiangsu Academy of Agricultural Sciences in Nanjing, Jiangsu Province, China. Three seedlings were planted per pot (20 cm × 30 cm), holding 6 kg of acid-washed pure quartz sand (pH 6.5), with the direct and daily irrigation of Hoagland and Arnon solution lacking nitrogen (N) [24]. Then, the seedlings at one month old were washed with water and transferred to the Hoagland and Arnon solution containing respective 0, 0.5, 1.0, 3.0, and 5.0 mM ammonium for water culture [12]. Each treatment was prepared with 3 biological replicates.

2.2. Morphological Measurement

Seven morphological indices of sweetpotato seedlings were measured in the root-swelling stage, including fresh weight, dry weight, leaf area index, fresh weight of storage root (SR), dry weight of SR, SR volume, and SR number [12]. The weight was measured using a PL202-L electronic balance (Mettler Toledo, Zurich, Switzerland). Leaf area index was estimated by a plant canopy analyzer LAI-2000 (LICOR Inc., Lincoln, NE, USA). The diameter was determined with a tape measure (Links Inc., Harbin, China). The volume was calculated by the water displacement method.

2.3. Measurements of Photosynthetic Parameters and Photosynthetic Product Contents

The leaves in the root-swelling stage were randomly selected and were exposed to 1 and 200 μmol·m−2·s−1 of PAR and 350 μL·L−1 of ambient CO2. The net photosynthesis rate (Pn), stomatal conductance (Gs), intercellular CO2 concentration (Ci), and transpiration rate (Tr) were measured using a Li-Cor 6400XT Portable Photosynthesis System (LICOR Inc., Lincoln, NE, USA) from 09:15 to 11:00 am on a sunny day. The recently expanded leaves in the root-swelling stage were selected for the measurement of chlorophyll (Chl) content, using a SPAD-502 Chl meter (Minolta corporation, Ramsey, NJ, USA). Three biological replications were carried out per sample.
The leaf, stem, and SR samples in the root swelling stage were collected from each treatment with three biological repeats, respectively. The contents of starch and sucrose were determined by dual-wavelength spectrophotometry and near-infrared (NIR) spectroscopy methods [25,26]. The standard starch used for the determination was produced from the Sigma company.

2.4. Hormone Content Determination

A total of 0.3 g of fresh SR in the root-swelling stage was ground in liquid nitrogen. ABA (abscisic acid), CK (cytokinin), and IAA (indole-3-acetic acid) were, respectively, extracted and purified following the procedures described by Qi et al. (2021) [27]. The extracts were analyzed with a high-performance liquid chromatography (HPLC) system at a UV wavelength of 254 nm. Sample separation was conducted using a C18 solid-phase extraction column.

2.5. Transcriptome Sequencing and Analysis

The SR samples in the root-swelling stage with treatments of 0 mM, 1 mM, and 5 mM were, respectively, prepared with three biological repeats for RNA-Sequencing using Illumina HiSeqTM 2000 150 PE. The reads with adaptors and/or uncertain bases more than 10% were deleted, as well as the low-quality reads of base quality ≤ 20 accounting for larger than 50%. The obtained high-quality clean reads were used to identify differentially expressed genes (DEGs) with log2|fold change| > 1 and the statistically significant value (p < 0.05) by DEGseq [28].
The screened DEGs by the gene ontology (GO) database (http://www.geneontology.org) were submitted to Blast2GO for enrichment analysis (accessed on 2 February 2025) [29]. To further identify the DEGs associated with carbohydrate allocation and plant hormone signal transduction, gene annotations were imported into the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.ad.jp/kegg/) (accessed on 4 February 2025). The p-value of GO terms and KEGG pathways was set at 0.05.

2.6. RT-qPCR Analysis

Four DEGs with significantly changed transcript abundances were selected to verify the accuracy of RNA-Seq by RT-PCR. A total of 1 μg of isolated RNA was used to proceed with first-strand cDNA synthesis using the PrimeScript™ RT reagent kit (Takara, Shanghai, China). The gene primers were designed by Primer Premier 5 (Table S1). The reference gene was the Actin gene. RT-qPCR was performed with a Strata gene Mx3000P qPCR System C1000 Thermal Cycler, containing 40 cycles of 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 30 s. Each amplification was repeated with three individual cDNA templates. Each treatment was prepared with three biological replicates. The relative expression level of the genes was calculated by the ΔΔCt method [30].

2.7. Statistics

A statistical analysis of morphological and physiological indices was carried out using Excel 2010 and SPSS 18.0. The variance was analyzed using one-way ANOVA, and multiple comparisons were performed by Duncan’s test (p < 0.05). The data in the figures and tables are the mean ± standard deviation.

3. Results

3.1. Morphological Traits of Sushu16 Seedlings in Root-Swelling Stage Under Different Ammonium Contents

The morphological results showed that the fresh weight, dry weight, and leaf area index of Sushu16 seedlings in the root-swelling stage exhibited an increasing trend with the increase in ammonium, reaching the maximum at 5 mM concentration. Meanwhile, the fresh weight, dry weight, volume, and number of SRs displayed a trend of a rapid rise and substantial decline, which peaked at 1 mM ammonium (Table 1). The significant values under 1 mM ammonium were 1.76, 1.74, 1.66, and 1.25 times higher than those under the control condition, respectively. This indicates that SR growth had no positive correlation with plantlet growth when challenged with high ammonium of >1 mM in sweetpotato in the root-swelling stage.

3.2. Photosynthetic Parameters in Sushu16 Seedlings in Root-Swelling Stage Under Different Ammonium Contents

Similarly, all the photosynthetic parameters of Sushu16 seedlings in the root-swelling stage displayed a rise–fall trend with the increase in ammonium, peaking at 1 mM ammonium. However, all the values exhibited a significant decrease under 5 mM ammonium. Even partial indicators were significantly lower than those under the control condition (Table 2). The chlorophyll content, photosynthetic rate (Pn), and stomatal conductance (Gs) under 1 mM ammonium treatment were significantly 1.30, 1.19-, and 1.85-fold higher than those under the control condition, respectively. However, there were no significant differences in the values of CO2 concentration (Ci) and transpiration rate (Tr). These phenomena indicated that 1 mM ammonium enhanced the photosynthesis of Sushu16 seedlings in the root-swelling stage.

3.3. Photosynthetic Products in Sushu16 Seedlings in Root-Swelling Stage Under Different Ammonium Contents

As shown in Figure 1, the sucrose contents in the leaves and stems were “down then up” with elevating ammonium concentration, which were lowest under 1 mM ammonium, while the starch content in SRs showed a trend of fall–rise, which was highest under 1 mM ammonium. The starch content in SRs in the root-swelling stage reached 7.1% at 1 mM ammonium content, which was 2.63-fold higher than that under the control condition. Accordingly, the contents of amylose and amylopectin in SRs displayed a similar tendency. The phenomenon demonstrated that 1 mM ammonium had a promotive effect on sucrose utilization and starch accumulation in Sushu16 seedlings in the root-swelling stage.

3.4. Hormone Contents in Sushu16 SRs in Root-Swelling Stage Under Different Ammonium Contents

As shown in Figure 2, the contents of cytokinin (CK), abscisic acid (ABA), and indole-3-acetic acid (IAA) in SRs in the root-swelling stage all increased first and then decreased, which peaked at 1 mM ammonium. The highest values were 878.67, 174.02, and 52.08 ng/g, which were significantly 1.56, 1.16, and 1.33 times higher than those under the control condition, respectively. This indicates that 1 mM ammonium had a stimulative influence on the hormone contents of Sushu16 SRs in the root-swelling stage.

3.5. Transcriptome Analysis of Sushu16 SRs in Root-Swelling Stage Under Different Ammonium Contents

3.5.1. The Screening of DEGs in Sushu16 SRs in the Root-Swelling Stage

Based on morphological and physiological responses, moderate ammonium (1.0 mM) significantly promoted starch accumulation in SRs, while excessive ammonium (5.0 mM) considerably reduced the starch content in SRs. Therefore, we conducted RNA-Seq of SR samples with respective treatments of 0, 1, and 5 mM ammonium to screen differentially expressed genes (DEGs) related to starch metabolism in Sushu16. Approximately 34.4, 32.5, and 31.7 million clean reads with an average length of 142 bp were gained from the RNA libraries of Sushu16 SRs with 0 mM, 1 mM, and 5 mM ammonium treatments, respectively. As shown in Figure 3, the hierarchical clustering of DEGs was grouped into 0 mM vs. 1 mM, 0 mM vs. 5 mM, and 1 mM vs. 5 mM horizontally, and five clusters longitudinally. A total of 3, 757 DEGs containing 2, 293 up-regulated genes (URGs) and 1, 464 down-regulated genes (DRGs) were obtained from the 0 mM vs. 1 mM comparison. As many as 7, 715 DEGs were screened from the 0 mM vs. 3 mM comparison, including 6, 957 URGs and 758 DRGs. The number of DEGs in 1 mM vs. 5 mM comparisons was 2, 525, containing 1, 499 URGs and 1, 026 DRGs. In the comparisons of 0 mM vs. 1 mM and 0 mM vs. 5 mM, the genes in clusters 1, 2, and 4 were mostly up-regulated, and the genes in clusters 3 and 5 were mostly down-regulated. Meanwhile, in the 1 mM vs. 5 mM comparison, the up-regulated and down-regulated genes were mixed in the different clusters.

3.5.2. GO Enrichment and KEGG Classification of DEGs in Sushu16 SRs in Root-Swelling Stage

According to GO enrichment, the DEGs were mainly distributed in the intracellular membrane-bound organelle, membrane-bound organelle, biological regulation process, stimulus response, biological regulation, nucleus, cellular regulation process, nucleobase-containing compound biosynthetic process, RNA biosynthetic process, and RNA metabolic process (Figure 4A). Based on the KEGG classification, the DEGs were mainly involved in three types of pathways including metabolism, environmental information processing, and organismal systems (Figure 4B). A large number of DEGs were classified into metabolism, mainly in carbon metabolism (536 genes), starch and sucrose metabolism (221 genes), carbon fixation in photosynthetic organisms (127 genes), N metabolism (83 genes), photosynthesis (76 genes), etc., followed by the pathway of environmental information processing, which contained 217 DEGs associated with plant hormone signal transduction. The last type was organismal systems, including the thyroid hormone signaling (52 genes), carbohydrate digestion and absorption (16 genes), phototransduction (16 genes), and phototransduction-fly pathways (14 genes).

3.5.3. The Identification of Significant DEGs Involved in Photosynthesis, Starch Metabolism, and Hormone Signal Transduction in Sushu16 SRs in the Root-Swelling Stage

A total of 34 significant DEGs involved in photosynthesis, starch metabolism, and hormone signal transduction were identified in SRs in the root-swelling stage with ammonium treatments (Excel S1, Table 3). As many as 10 DEGs were identified to be involved in photosynthetic metabolism. Eight out of them were all up-regulated in the ammonium trials. Only one Zinc finger protein ZAT10 with an ID of c67603_g1 was down-regulated in the three comparisons. The DEG with an ID of c59492_g1 was up-regulated in the comparisons of 0 mM vs. 1 mM and 0 mM vs. 5 mM, while it was down-regulated in the 1 mM vs. 5 mM comparison, which was associated with carbon fixation in photosynthetic organisms. Among the 19 DEGs annotated to participate in starch biosynthetic process, two DEGs were down-regulated, while the other 17 genes were all up-regulated in all comparison groups. There were five hormone-related DEGs under the ammonium stimulus, including four up-regulated genes and one down-regulated gene. Particularly, four out of them were involved in the auxin-mediated signaling pathway.

3.6. RT-qPCR Validation of Significant DEGs

To validate the data accuracy of RNA-Seq, we selected four significant DEGs involved in photosynthesis and starch metabolism for RT-qPCR. The results indicated that the relative expression levels of c63371_g1, c54445_g1, c73282_g1, and c73484_g1 under 1 mM ammonium were 2.51-, 7.32-, 1.85-, and 1.87-fold higher than those under the control condition, respectively. The values in the 5 mM ammonium trial were, respectively, 4.59-, 9.65-, 4.31-, and 6.49-fold, compared to the control condition. The relative expression changes of these significant DEGs quantified by RT-qPCR were generally consistent with mRNA abundance alternations profiled with RNA-Seq under different ammonium contents (Figure 5, Table 3). The results indicated that the ammonium stimulus induced an up-regulation of these genes to promote photosynthesis and starch accumulation in Sushu16.

4. Discussion

Ammonia nitrogen (N) application has been reported to greatly influence nutrient partitioning and root growth, particularly in the tuberous crops where assimilate translocation is critical for yield formation [19,31]. Sweetpotato has a typical sink–source relationship, as a type of tuberous root crop [8]. In this study, we investigated the morphological, physiological, and molecular responses of the Sushu16 variety in the root-swelling stage with treatments of different ammonium contents (0, 0.5, 1.0, 3.0, and 5.0 mM) to trace the specific mechanisms of action of ammonium application on assimilate translocation and SR growth in sweetpotato.

4.1. The Effects of Ammonium Application on Source–Sink Conversion and SR Production in Sweetpotato

Morphological indicators indicated that the moderate ammonium (1 mM) supply significantly improved SR production, while excessive ammonium (5 mM) considerably reduced SR biomass in Sushu16 transplants, which is consistent with previous results showing that both N deficiency and N toxicity inhibit the SR production of sweetpotato [11,13]. Chen et al. (2015) confirmed that 60 kg/ha of N fertilizer contributes to the optimal SR yield as well starch quality in the treatments with five N gradients (0, 60, 120, 180, and 240 kg/ha) in Xushu 28 and Xushu 22 cultivars [13]. With additional N fertilizer treatments of 0, 15, and 30 kg/ha, the starch size of the Sushu16 variety first increased and then decreased [14]. The root dry matter of Yanshu25 and Shangshu19 first increased and then decreased under five N fertilizers (0, 60, 120, 180, and 240 kg/ha), peaking in the 120 kg/ha trial [11]. Restricted root growth and leaf chlorosis are primary phenotypic indicators of ammonium toxicity in plants [19], which is in agreement with the present results showing that the SR biomass and chlorophyll content in Sush16 were significantly decreased under 5 mM ammonium.
The physiological responses to ammonium were also striking in Sushu16 seedlings. The significant improvement in photosynthetic efficiency and chlorophyll content in Sushu16 under 1 mM ammonium aligns with the previous report showing that moderate ammonium increased photosynthesis and plant biomass in maize [32]. The significant increase in stomatal conductance (Gs) with 1 mM ammonium further supports the fact that ammonium at optimal concentrations facilitates gas exchange, potentially leading to enhanced photosynthesis [33]. However, excessive ammonium can lead to a lower chlorophyll content and declined photosynthetic rate in crops [19]. This conforms to the present results showing that 5 mM ammonium significantly reduced all photosynthetic parameters in Sushu16 transplants. N deficiency and N excess can, respectively, cause insufficient growth and the overgrowth of source leaves, thereby leading to an imbalance in the source–sink relationship and production reduction in sweetpotato [12]. An optimal ammonium treatment can enhance sucrose-to-hexose conversion via invertase activation, channeling carbon resources into cell division processes that drove SR growth in sweetpotato [16]. The concurrent decrease in sucrose content in the leaf and stem, coupled with a peak of starch accumulation in SR under 1 mM ammonium, revealed that moderate ammonium promoted the source–sink conversion of sweetpotato, which conforms to previous results [12,16], while high ammonium application led to decreased starch accumulation in SRs, which accords with the conclusion that excess ammonium likely induced metabolic and osmotic imbalances and nutrient absorption disturbances that impair phloem loading or trigger compensatory sucrose retention in leaves [19,34].

4.2. The Effects of Ammonium Application on SR Hormone Contents in Sweetpotato

Increasing evidence supports the existence of a feed-forward loop between N signaling and hormonal status [35,36]. N supply regulates plant growth and development at several levels of integration through the control of hormone synthesis, transport, and signaling [36]. For example, high nitrate supply induces a systemic inhibitory effect on lateral root development in Arabidopsis, which is mediated by ABA [37]. N supply enhances leaf growth via increasing auxin synthesis to build a large sink for carbon and N utilization in maize [32]. On the other hand, hormonal signals play an essential role in regulating plant growth and development and influencing various physiological processes such as N-sensing, -uptake, and -assimilation [35,36]. For instance, ABA is known to enhance sink strength by stimulating sucrose transporters [38], while CK and IAA synergistically regulate the cell division and expansion of young roots, thereby promoting SR formation [16,39].
The coordination of N signaling and hormonal contents orchestrates profound developmental modifications in plants, with particularly pronounced effects on root architecture [35]. SR swelling is a complex process characterized by the massive deposition of starch and storage proteins, which eventually results in its enlargement in sweetpotato [8]. The processes are mediated by the interaction of key phytohormones such as ABA, CK, and IAA and coordinated by the expression of relevant genes [6,40]. N deficiency can result in an insufficient accumulation of sucrose and starch through the inhibition of the simultaneous decrease in phytohormone levels in crops [41]. In this study, the peak in the relative contents of ABA, CK, and IAA and starch content in Sushu16 SRs with 1 mM ammonium treatment suggested that moderate ammonium regulated the phytohormone balance to promote SR swelling in sweetpotato. This has also been supported by previous findings showing that optimal ammonium treatment produced the highest SR yield and number per plant by modulating the concentrations of ZR and IAA in potential SRs of two sweetpotato cultivars [16]. In contrast, the decline in hormone contents at high ammonium levels reflects the stress-induced suppression of hormonal biosynthesis, as excessive ammonium can trigger reactive oxygen species (ROS) accumulation and cause carbon scarcity in plants [17,42]. This is in agreement with the present results showing that the relative contents of ABA, CK, and IAA were all considerably reduced with the treatment of 5 mM ammonium in Sush16.

4.3. The Effects of Ammonium Application on Transcriptional Alterations in Sweetpotato

Alterations in gene expression linked to nutrient status can lead to modified metabolic responses, further emphasizing the importance of genetic regulation in nutrient uptake and utilization in plants [43]. Ammonium can trigger rapid and specific changes in cytosolic pH, redox status, gene expression, post-translational modifications of proteins, metabolism, and other physiological and molecular responses [17]. There has been increasing evidence showing that an ammonium stimulus can induce tremendous alterations in gene expression to induce adequate metabolic adjustments. For instance, substantial transcriptional differences were pronounced in the gene modules of photosynthesis, mitochondrial metabolism, ammonium metabolism, and cell wall biosynthesis in the (NH4)2SO4-grown Arabidopsis, leading to nutritional and metabolic imbalances [23]. In rice, all the DEGs associated with carbohydrate and amino acid metabolisms were up-regulated in shoots under high ammonium supply, implying improved carbohydrate and N metabolisms [44]. The spatiotemporal specificity of gene expression profiles in rice under excess ammonium reveals that the up-regulated hormone-associated DEGs contribute to the coordinated regulation of phytohormones. As well, the up-regulated DEGs mainly participating in phenylpropanoid and amino acid metabolism lead to key adjustments of energy metabolism [45].
Several studies have confirmed that moderate ammonium application contributes to yield formation in different sweetpotato cultivars [11,13,16]. However, the underlying mechanisms of the transcriptional alterations of ammonium application on assimilate translocation and SR growth are not well understood in sweetpotato. In this study, gene profiling provided further insights into molecular mechanisms activated by the ammonium stimulus in the Sushu16 cultivar. As many as 536, 221, 127, and 76 screened DEGs were, respectively, classified into carbon metabolism, starch and sucrose metabolism, carbon fixation in photosynthetic organisms, and photosynthesis in the ammonium trials. Notably, transcriptomic analysis revealed a total of 29 significant DEGs related to photosynthesis and starch and sucrose metabolism, which aligns with the previous studies showing that N signaling directly regulates carbon enzyme activity via transcriptional control [46,47]. With ammonium treatment, the significantly differential expression of 10 photosynthetic genes accorded with the increase in Pn. One significant photosynthetic DEG with an ID of c59492_g1 was highly homologous with phosphoenolpyruvate carboxylase 3 in Arabidopsis, which has been reported to form oxaloacetate through the carboxylation of phosphoenolpyruvate [48]. In particular, it was up-regulated in the comparisons of 0 mM vs. 1 mM and 0 mM vs. 5 mM, while it was down-regulated in the comparison of 1 mM vs. 5 mM. The expression pattern of the gene further supports the fact that 1 mM ammonium was optimal in promoting photosynthesis in sweetpotato. Similarly, significant expression changes in as many as 19 starch-metabolism-related DEGs likely drove starch biosynthesis in Sushu16, such as starch synthase, starch branching enzyme, and glucose-1-phosphate adenylyltransferase [7]. This accords with the result that ammonium influences key metabolic pathways associated with carbohydrate allocation in sweetpotato [49]. Furthermore, the screening of 217 DEGs enriched in “plant hormone signal transduction” terms was consistent with plant hormone changes in SRs with ammonium treatments. Particularly, the identification of four significant DEGs involved in the auxin-mediated signaling pathway conformed to the SR growth phenotype of Sushu16. For example, the significant DEGs with IDs of c66686_g1 and c71847_g1 are highly homologous to auxin response factors, ARF8/ARF6, which were reported to positively regulate adventitious rooting in Arabidopsis [50,51]. AUX/IAA transcriptional regulator, SHY2, has high sequence identification with the significant DEG with an ID of c69629_g2, indicating its role in regulating lateral root formation [52]. To sum up, moderate ammonium application induces striking alterations in gene expression, contributing to efficient source–sink conversion and improved SR production in sweetpotato.

5. Conclusions

In this study, we investigated the morphological, physiological, and molecular effects of applying varying amounts of ammonium on assimilate translocation and the storage root (SR) growth of the Sushu16 variety in the root-swelling stage. The plant weight and leaf area index of Sushu16 seedlings exhibited a rise trend with the increase in ammonium, while SR biomass displayed a fall–rise pattern, which peaked at 1 mM ammonium. Similarly, photosynthesis indicators and hormonal contents in SRs reached optimum values under 1 mM ammonium. It is noteworthy that the dynamic interplay between sucrose depletion in source organs and starch accumulation in SRs highlights the positive effects of 1 mM ammonium on modulating an efficient sucrose–starch conversion. Furthermore, the morphological and physiological responses were supported by the profiling of numerous DEGs participating in photosynthesis, starch metabolisms, and hormone signal transduction. Of particular importance was the identification of 34 significant DEGs involved in photosynthesis (10 genes), starch and sucrose metabolism (19 genes), and the hormone signal pathway (5 genes), conforming to the enhanced photosynthetic rate, efficient source–sink conversion, and improved hormone contents in SRs. All the results indicated that a dose of 1 mM ammonium had a promotive effect on the growth and production of SR in Sushu16, which can be applied to foliar fertilization in the root-swelling stage to maximize the yield of sweetpotato, reducing the cost and avoiding the growth inhibition of excessive nitrogen fertilizer.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15061272/s1, Table S1: Primers used for RT-qPCR; Excel S1: Significant DEGs related to photosynthesis, starch and sucrose metabolism, and hormone signal pathway.

Author Contributions

Conceptualization, W.Y.; Methodology, W.Y.; software, W.Y.; validation, R.Z. and Q.T.; formal analysis, R.Z. and Q.T.; investigation, R.Z. and Q.T.; resources, C.C., C.Z., W.S. and C.L.; data curation, C.L.; writing—original draft preparation, W.Y.; writing—review and editing, C.L.; supervision, C.C., C.Z., W.S. and C.L.; project administration, C.C., C.Z., W.S. and C.L.; funding acquisition, C.L. and W.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Jiangsu Agricultural Science and Technology Innovation Fund (CX(22)3049), and the Municipal Level Basic Research Program of Huai’an City (HABL2023053).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article, and further inquiries can be directed at the corresponding author.

Acknowledgments

The authors thank Jianping Wang from the University of Florida for the critical reading and editing of the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Photosynthetic products in Sushu16 seedlings in the root-swelling stage under different ammonium contents. (A) Sucrose contents in the leaves and stems. White columns and gray columns represent the sucrose contents in the leaves and stems, respectively. (B) The contents of amylose, amylopectin, and starch in SRs. Gray columns, black columns, and white columns represent the contents of amylose, amylopectin, and starch in SRs, respectively. Different lowercase letters in the same column indicate significant differences among the treatments (p < 0.05).
Figure 1. Photosynthetic products in Sushu16 seedlings in the root-swelling stage under different ammonium contents. (A) Sucrose contents in the leaves and stems. White columns and gray columns represent the sucrose contents in the leaves and stems, respectively. (B) The contents of amylose, amylopectin, and starch in SRs. Gray columns, black columns, and white columns represent the contents of amylose, amylopectin, and starch in SRs, respectively. Different lowercase letters in the same column indicate significant differences among the treatments (p < 0.05).
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Figure 2. Hormone contents of Sushu16 SRs in the root-swelling stage under different ammonium contents. Different lowercase letters in the same column indicate significant differences among the treatments (p < 0.05). White columns, black columns, and gray columns represent the contents of ABA (abscisic acid), IAA (indole-3-acetic acid), and CK (cytokinin) in SRs under different ammonium contents, respectively.
Figure 2. Hormone contents of Sushu16 SRs in the root-swelling stage under different ammonium contents. Different lowercase letters in the same column indicate significant differences among the treatments (p < 0.05). White columns, black columns, and gray columns represent the contents of ABA (abscisic acid), IAA (indole-3-acetic acid), and CK (cytokinin) in SRs under different ammonium contents, respectively.
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Figure 3. A heatmap of DEGs in Sushu16 SRs in the root-swelling stage. Red represents UEGs and green represents DEGs. The colorful vertical bars denote different gene clusters.
Figure 3. A heatmap of DEGs in Sushu16 SRs in the root-swelling stage. Red represents UEGs and green represents DEGs. The colorful vertical bars denote different gene clusters.
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Figure 4. GO enrichment and KEGG analysis of DEGs in Sushu16 SRs under different ammonium contents. (A) GO enrichment; (B) KEGG classification.
Figure 4. GO enrichment and KEGG analysis of DEGs in Sushu16 SRs under different ammonium contents. (A) GO enrichment; (B) KEGG classification.
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Figure 5. The relative expression levels of four significant DEGs quantified by RT-qPCR. White columns and black columns represent the relative expression levels of the genes quantified by RT-qPCR in SRs with treatments of 1 mM and 5 mM ammonium, respectively. The dotted line and dashed line represent the mRNA abundance alternations of the genes profiled by RNA-Seq in SRs with treatments of 1 mM and 5 mM ammonium, respectively.
Figure 5. The relative expression levels of four significant DEGs quantified by RT-qPCR. White columns and black columns represent the relative expression levels of the genes quantified by RT-qPCR in SRs with treatments of 1 mM and 5 mM ammonium, respectively. The dotted line and dashed line represent the mRNA abundance alternations of the genes profiled by RNA-Seq in SRs with treatments of 1 mM and 5 mM ammonium, respectively.
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Table 1. Morphological traits of Sushu16 seedlings in root-swelling stage under different ammonium contents.
Table 1. Morphological traits of Sushu16 seedlings in root-swelling stage under different ammonium contents.
Morphological Indices0 mM NH40.5 mM NH41 mM NH43 mM NH45 mM NH4
Fresh weight (g)15.09 ± 2.44 c16.88 ± 1.34 c18.6 ± 2.34 bc22.64 ± 2.49 b32.15 ± 5.73 a
Dry weight (g)2.71 ± 0.44 c2.98 ± 0.24 c3.32 ± 0.42 bc3.96 ± 0.43 b5.54 ± 0.99 a
SR fresh weight (g)12.78 ± 2.23 b16.12 ± 2.86 ab22.55 ± 3.56 a15.77 ± 3.17 b6.17 ± 1.08 c
SR dry weight (g)3.21 ± 0.56 b4.07 ± 0.72 ab5.59 ± 0.88 a3.96 ± 0.79 b1.58 ± 0.27 c
SR volume (cm3)131.75 ± 16.64 b169.68 ± 19.55 ab219.89 ± 43.47 a162.57 ± 31.54 ab73.61 ± 11.46 c
SR number1.33 ± 0.47 ab1.66 ± 0.54 a1.85 ± 0.32 a1.63 ± 0.35 ab1 ± 0.12 b
Leaf area index2.84 ± 0.34 b3.14 ± 0.42 b3.67 ± 0.48 ab3.73 ± 0.53 ab4.25 ± 0.62 a
Notes: Different lowercase letters in the same column indicate a significant difference among the treatments (p < 0.05).
Table 2. Photosynthetic parameters of Sushu16 seedlings in root-swelling stage under different ammonium contents.
Table 2. Photosynthetic parameters of Sushu16 seedlings in root-swelling stage under different ammonium contents.
Photosynthetic Parameters0 mM NH40.5 mM NH41 mM NH43 mM NH45 mM NH4
Chlorophyll content12.00 ± 1.54 b13.10 ± 2.62 ab15.63 ± 1.34 a12.40 ± 3.12 ab10.33 ± 2.99 b
Pn [μmol m−2 s −1]9.88 ± 0.25 b10.52 ± 0.48 ab11.75 ± 1.54 a10.11 ± 2.26 ab8.33 ± 1.32 b
Gs [μmol m−2 s −1]0.108 ± 0.011 b0.151 ± 0.025 ab0.200 ± 0.029 a0.143 ± 0.039 ab0.053 ± 0.006 c
Ci [μmol mol −1]212.5 ± 25.63 a229.0 ± 27.49 a234.5 ± 31.77 a213.0 ± 24.68 a108.5 ± 12.35 b
Tr [μmol m−2 s −1]4.33 ± 0.58 ab4.91 ± 0.31 ab5.50 ± 0.37 a4.57 ± 0.62 b2.11 ± 0.15 c
Notes: Different lowercase letters in the same column indicate significant differences among the treatments (p < 0.05).
Table 3. Significant DEGs involved in photosynthesis, starch metabolic pathways, and hormone signal transduction in Sushu16 SRs in root-swelling stage under different ammonium contents.
Table 3. Significant DEGs involved in photosynthesis, starch metabolic pathways, and hormone signal transduction in Sushu16 SRs in root-swelling stage under different ammonium contents.
Gene ID1 mM vs. 5 mM
(Up/Down)		0 mM vs. 1 mM
(Up/Down)		0 mM vs. 5 mM
(Up/Down)		Gene AnnotationMetabolic Pathways
c59492_g1Down(−1.81)Up(4.40)Up(2.58)Phosphoenolpyruvate carboxylase 3Photosynthesis; carbon fixation in photosynthetic organisms
c63371_g1Up(1.61)Up(3.27)Up(4.87)Chlorophyll a-b binding protein CP24 10APhotosynthesis—antenna proteins
c67118_g1Up(1.61)Up(2.09)Up(3.70)Solanesyl diphosphate synthase 2Photosynthesis
c67603_g1Down(−1.04)Down(−2.85)Down(−3.89)Zinc finger protein ZAT10Photoprotection; photosynthesis
c67605_g1Up(1.25)Up(1.76)Up(3.01)Serine/threonine-protein kinase STN7Regulation of photosynthesis; chloroplast thylakoid membrane
c67668_g1Up(1.54)Up(3.18)Up(4.13)Chlorophyll a-b binding protein 40Photosynthesis—antenna proteins
c68701_g1Up(1.78)Up(2.28)Up(4.05)Magnesium-protoporphyrin IX monomethyl ester [oxidative] cyclasePhotosynthesis; chlorophyll biosynthetic process
c73267_g1Up(2.34)Up(2.28)Up(4.63)Chlorophyll a-b binding protein; ethylene-responsive elongation factor EF-Ts precursorPhotosynthesis—antenna proteins
c74975_g1Up(1.13)Up(2.21)Up(3.34)Phosphoenolpyruvate carboxylase 4Photosynthesis; carbon fixation in photosynthetic organisms
c75565_g1Up(1.12)Up(1.37)Up(2.48)Magnesium-chelatase subunit ChlI, chloroplastic Chlorophyll biosynthetic process
c54445_g1Up(2.01)Up(8.15)Up(10.15)Glucose-1-phosphate adenylyltransferase large subunitStarch biosynthetic process; glycogen biosynthetic process
c55971_g1Up(1.32)Up(2.26)Up(3.58)Pyruvate phosphate dikinaseStarch catabolic process; carbohydrate metabolic process
c62758_g1Down(−4.34)Down(−2.30)Down(−6.64)Starch branching enzyme I Starch biosynthetic process
c62840_g1Down(−2.75)Down(−2.98)Down(−5.73)PfkB family carbohydrate kinaseStarch and sucrose metabolism
c67608_g1Up(1.45)Up(1.96)Up(3.41)Granule-bound starch synthase 2Starch synthase activity; starch biosynthetic process
c68645_g4Up(1.12)Up(2.10)Up(3.22)Phosphoglucan phosphatase DSP4Starch catabolic process; carbohydrate phosphatase activity
c70089_g2Up(1.18)Up(4.16)Up(5.34)Isoamylase 2Starch biosynthetic process
c71691_g1Up(1.55)Up(1.66)Up(3.21)Starch branching enzyme IIStarch and sucrose metabolism
c73282_g1Up(2.09)Up(1.55)Up(3.64)Glucose-1-phosphate adenylyltransferase small subunitStarch biosynthetic process; glycogen biosynthetic process
c73484_g1Up(3.57)Up(4.47)Up(8.04)Pectinesterase/pectinesterase inhibitorStarch and sucrose metabolism
c74637_g1Up(1.59)Up(3.82)Up(5.41)Isoamylase 1Starch biosynthetic process
c74946_g4Up(2.41)Up(1.99)Up(4.40)Phosphoribosyl transferase 2Starch and sucrose metabolism; Pentose and glucuronate interconversion
c74989_g1Up(2.27)Up(2.03)Up(3.35)carbohydrate-binding module (CBM20)Starch catabolic process; starch binding
c76971_g1Up(1.10)Up(3.09)Up(4.19)1,4-alpha-glucan branching enzyme/starch branching enzyme IICarbohydrate transport and metabolism
c77775_g1Up(1.09)Up(2.26)Up(3.36)4-alpha-glucanotransferaseStarch catabolic process; starch binding
c78303_g2Up(1.29)Up(1.19)Up(2.18)Isoamylase 3Carbohydrate metabolic process; starch catabolic process
c79207_g2Up(1.72)Up(2.53)Up(4.25)Granule-bound starch synthase IStarch biosynthetic process
c79256_g2Up(1.60)Up(1.85)Up(3.45)Glucose-1-phosphate adenylyltransferaseStarch biosynthetic process; glycogen biosynthetic process
c79783_g1Up(1.28)Up(1.36)Up(2.64)PPDK_N, Pyruvate phosphate dikinaseStarch catabolic process; carbohydrate kinase activity
c51798_g1Up(2.08)Up(2.53)Up(4.61)Auxin-responsive protein, IAA16Plant hormone signal transduction
c66686_g1Up(1.26)Up(2.98)Up(4.24)Auxin response factor, ARF8Auxin-mediated signaling pathway
c69629_g2Down(−2.08)Down(−2.22)Down(−4.30)AUX/IAA transcriptional regulator, SHY2Auxin-mediated signaling pathway
c71847_g1Up(1.19)Up(2.22)Up(3.41)Auxin response factor, ARF6Auxin-mediated signaling pathway
c78272_g2Up(1.31)Up(1.21)Up(2.52)Rapid ALkalinization Factor 31Hormone activity
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Yao, W.; Zhou, R.; Tan, Q.; Zhuang, C.; Shao, W.; Chen, C.; Li, C. Effects of Ammonium on Assimilate Translocation and Storage Root Growth in Sushu16 in Root-Swelling Stage. Agronomy 2025, 15, 1272. https://doi.org/10.3390/agronomy15061272

AMA Style

Yao W, Zhou R, Tan Q, Zhuang C, Shao W, Chen C, Li C. Effects of Ammonium on Assimilate Translocation and Storage Root Growth in Sushu16 in Root-Swelling Stage. Agronomy. 2025; 15(6):1272. https://doi.org/10.3390/agronomy15061272

Chicago/Turabian Style

Yao, Wenjing, Rui Zhou, Qin Tan, Chun Zhuang, Wenqi Shao, Chuan Chen, and Chuanzhe Li. 2025. "Effects of Ammonium on Assimilate Translocation and Storage Root Growth in Sushu16 in Root-Swelling Stage" Agronomy 15, no. 6: 1272. https://doi.org/10.3390/agronomy15061272

APA Style

Yao, W., Zhou, R., Tan, Q., Zhuang, C., Shao, W., Chen, C., & Li, C. (2025). Effects of Ammonium on Assimilate Translocation and Storage Root Growth in Sushu16 in Root-Swelling Stage. Agronomy, 15(6), 1272. https://doi.org/10.3390/agronomy15061272

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