Exploring the Molecular Landscape of Nitrogen Use Efficiency in Potato (Solanum tuberosum L.) under Low Nitrogen Stress: A Transcriptomic and Metabolomic Approach

Abstract

Enhancing crop nitrogen use efficiency (NUE) in agricultural sciences is a pivotal challenge, particularly for high-demand crops like potatoes (Solanum tuberosum L.), the world’s third most significant food crop. This study delves into the molecular responses of potatoes to low nitrogen (LN) stress, employing an integrative approach that combines transcriptomics and metabolomics to compare two cultivars with divergent NUE traits: XS6, known for its high NUE, and NS7, characterized by lower NUE. Our research unveils that XS6 exhibits higher chlorophyll and N content, increased tuber yield, and elevated N assimilation capacity under LN stress conditions compared to NS7. Through transcriptome analysis, we identified critical genes involved in C and N metabolism that had higher expression in XS6. A significant discovery was the high-affinity nitrate transporter 2.7 gene, which showed elevated expression in XS6, suggesting its key role in enhancing NUE. Metabolomics analysis further complemented these findings, revealing a sophisticated alteration of 1252 metabolites under LN stress, highlighting the dynamic interplay between carbon and N metabolism in coping with N scarcity. The integration of transcriptomic and metabolomic data underscored the crucial role of trehalose in mitigating N deficiency and enhancing NUE. This study provides novel insights into the molecular mechanisms governing NUE in potatoes, offering valuable perspectives for molecular breeding to enhance NUE in potatoes and potentially other crops.

1. Introduction

Nitrogen (N), an elemental cornerstone for the growth and development of plants [1,2], is intricately involved in the biosynthesis of vital molecules including nucleic acids, proteins, chlorophyll, alkaloids, vitamins, and hormones [3,4]. The assimilation of sufficient N, available in the soil primarily in inorganic (e.g., nitrate and ammonium) and organic (e.g., urea, amino acids, and peptides) forms, is crucial for optimal plant growth [5,6,7]. Despite its abundance, the limited availability of these forms of N often restricts crop yields, making the judicious use of N fertilizers in agriculture indispensable. To increase crop yields, farmers have resorted to excessive chemical N fertilizer application in most agricultural systems [8]. However, the prevalent over-application of chemical nitrogen fertilizers in agricultural systems escalates production costs and imposes severe environmental challenges. Despite its substantial application, only 30–50% of the applied N fertilizer is effectively taken up by crops, with the remainder contributing to atmospheric pollution through nitrous oxide emissions—a potent greenhouse gas—or exacerbating eutrophication through leaching into water systems [9,10,11]. Amidst growing global food demands driven by an ever-increasing population, developing crop genotypes characterized by enhanced nitrogen use efficiency (NUE) presents a promising strategy [12]. Such genotypes offer the potential to optimize N uptake and utilization, thereby mitigating the environmental impact associated with N fertilizers while simultaneously improving agricultural productivity [13]. This introduction sets the stage for our investigation into the roles and regulatory mechanisms underlying NUE in plants, highlighting the significance of N in plant biology and the imperative for sustainable agricultural practices.

NUE is a complex trait influenced by genetic and environmental factors, encompassing processes like N uptake, assimilation, and remobilization [14]. Cereal crops, for instance, often define NUE in terms of grain yield relative to the amount of N fertilizer applied, a measure that has guided research and breeding efforts for decades [15,16]. An ideal nitrogen-efficient genotype should have both high nitrogen use efficiency and strong uptake efficiency [17]. Therefore, it is necessary to conduct a comprehensive evaluation based on multiple nitrogen efficiency indicators, which will help in selecting genotypes that take into account both nitrogen absorption and utilization. Recent advancements in the understanding of genes involved in N transport and metabolism have shed light on the mechanisms that facilitate N utilization across diverse plant species, including Arabidopsis [1,18], rice (Oryza sativa L.) [19,20], cotton (Gossypium L.) [21], and maize (Zea mays L.) [22], paving the way for targeted genetic interventions to enhance NUE. Despite progress in identifying N-responsive genes and regulatory networks that modulate plant growth in response to N availability, the molecular bases of N sensing and signaling remain largely unexplored, representing a critical gap in improving NUE [23,24]. This gap underscores the ongoing challenge and the need for continued investigation into the molecular underpinnings of NUE to unlock new pathways for enhancing the efficiency with which crops utilize N.

The potato (Solanum tuberosum L.), ranked as the third most crucial food crop globally following rice and wheat, plays a pivotal role in ensuring food, nutritional, and economic security worldwide [25,26]. Nitrogen profoundly influences potato plant growth, tuber yield, and quality. Potatoes are highly input-intensive, requiring substantial N fertilization for satisfactory yields. However, only 40–50% of the applied N is effectively utilized by the potato plants, with the excess contributing to environmental concerns such as groundwater contamination [27]. Developing potato cultivars with enhanced NUE is therefore critical for reducing reliance on N fertilizers and promoting sustainable agricultural practices. Additionally, wild potato germplasm outperformed common cultivars in terms of NUE, particularly S. chacoense, S. commersonii, S. kurtzianum, S. microdontum, and S. phureja, which had equal to or greater dry weights than the cultispecies [28,29,30,31]. It has been proposed that the high-affinity nitrate transporter in potato roots is a promising candidate gene for manipulating nitrogen absorption and transport. By utilizing CRISPR/Cas9 and other fundamental editing techniques, it becomes feasible to improve the efficiency of nitrogen utilization in plants [32]. These investigations contribute to comprehending the genetic foundation of potato NUE and offer genetic insights for developing potato varieties with enhanced nitrogen use efficiency.

The integration of transcriptomic and metabolomic analyses offers a promising avenue for dissecting the complex biological systems of plants. Such comprehensive approaches have already yielded significant insights into plant responses to various stresses, including salinity in lyceum, drought in switchgrass, and color shifts in Lilium bulbs, demonstrating their utility in plant science research [33,34,35]. Despite these advancements, there remains a scarcity of information on the specific genes that contribute to improving NUE in potatoes. Addressing this gap is crucial for advancing our understanding of N management in potato cultivation and developing strategies to enhance agricultural sustainability.

In the present study, we explored the metabolic pathways and regulatory networks mediating low N signal transduction in potatoes, employing both transcriptomics and metabolomics. Focusing on the leaves and roots 45 days after planting, we assessed the plants under low and standard N application conditions. The objective was to delineate the adaptive response mechanisms of potatoes to varying N levels, with a particular emphasis on leaf and root dynamics in nutrient utilization. Through this integrated analytical approach, our study aims to shed light on the intricate molecular dialogues that govern N utilization in potatoes, potentially paving the way for advances in crop management and genetic improvement strategies.

2. Materials and Methods

2.1. Plant Material and Treatment

This study was conducted at the Experimental Base of the Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences in Hohhot, Inner Mongolia, China (40°77′ N, 111°67′ E) during the 2019–2021 potato growing season. The basic soil nutrient levels in the 0–20 cm layer are presented in Table S1. A split-plot design was implemented for the field experiments, with N treatments forming the main plots. Urea (46.0% N) was used to fertilize plants at total N supply rates of 0 kg N·hm⁻² (no N fertilizer, control), 150 kg N·hm⁻² (low N, LN), and 300 kg N·hm⁻² (normal N, NN) [36]. The potato cultivars used as subplots included the N-efficient genotype Xisen 6 (XS6) and the N-inefficient genotype Neishu 7 (NS7), which differ in yield potential but share a similar flowering period (Table S2). Each plot was 27 m² in size, consisting of 5 rows with each row being 6 m long and spaced 0.9 m apart, resulting in a density of 45,000 plants·hm⁻². Approximately two-thirds of the N fertilizer was applied in solid form to the soil before plowing and planting, together with 180 kg·hm⁻² P₂O₅ and 300 kg·hm⁻² K₂O. The remaining one-third of the nitrogen fertilizer was applied via fertigation during the seedling stage. The precipitation recorded during the growing seasons from 2019 to 2021 was 363.6 mm, 343.5 mm, and 326.3 mm, respectively. The mean temperatures for these years were correspondingly measured at 19.12 °C, 18.35 °C, and 19.21 °C (Figure S1). Plants were watered 8 times (225 m³·hm⁻² of water each time) using a drip irrigation system during the growth period.

2.2. Analysis of Growth and Yield Parameters

Our study conducted a comprehensive analysis to assess various growth and yield parameters of potato plants, focusing on the crucial developmental stages (tuber expansion stage and tuber maturity stage) at 45 and 85 days after planting (DAP). The nitrogen efficiency-related indicators for the potato varieties XS6 and NS7 were evaluated during the tuber maturity stage (85 DAP) in 2019–2020. Based on two years of evaluation, transcriptomic and metabolomic studies were conducted in 2021. We measured the chlorophyll content, the total N content of the entire plant, and the activity of key enzymes involved in N metabolism at the tuber expansion stage (45 DAP). Concurrently, evaluations of tuber yield and NUE parameters were systematically performed at the tuber maturity stage (85 DAP).

The leaf discs were extracted using 25 mL of 95% ethanol and soaked for 48 h. The concentrations of Chl a and b were respectively measured at 649 nm and 665 nm with a spectrophotometer (Shimadzu, UV-1800, Kyoto, Japan). For total N content analysis, we processed samples, including leaf, stem, root, and tuber, collected at both 45 DAP and 85 DAP. These samples were initially rinsed with deionized water, then dried in an oven set at 105 ℃ for 30 min and maintained at 80 ℃ until a constant weight was achieved. Post-drying, the samples were pulverized into powder and digested using an H₂SO₄-H₂O₂ mixture [37]. N concentration was determined with a continuous flow analyzer (AA3, Seal Analytical Inc., Southampton, UK).

Enzymatic activity measurements for nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (Fd-GOGAT and NADH-GOGAT), and glutamate dehydrogenase (GDH) were conducted using assay kits supplied by Suzhou Grace Biotechnology Co., Ltd., China, designated as G0402W96 for NR, G0401W96 for GS, G0404W for Fd-GOGAT, G0403W for NADH-GOGAT, and G0405W for GDH, respectively.

Tuber yield, expressed in tonnes per hectare (t·hm⁻²), was accurately measured at tuber maturity stage (85 DAP) by selecting the central two rows from each plot. NUE was calculated as the quotient of plant dry matter accumulation over the total N applied as fertilizer, employing the formula outlined by Moll et al. [15] and Zebarth et al. [30].

2.3. RNA Extraction, Library Construction, RNA Sequencing, and Data Analysis

The transcriptomic analysis focused on the fourth leaf and root from the top of the potato plants, collected at 45 DAP during the 2021 growing season. RNA sequencing (RNA-seq) was employed to examine the transcriptomic profiles of these samples from the two distinct potato cultivars at this developmental stage. Total RNA was extracted from leaf and root samples using the RNAprep Pure Plant Kit (DP441, Tiangen, Beijing China), following a protocol to ensure the comprehensive capture of the plant’s transcriptomic landscape.

The enrichment of mRNA, specifically those transcripts containing polyA tails, was achieved through oligo (dT) beads, a critical step in ensuring the quality and specificity of the RNA samples for sequencing. The construction of the cDNA library, a pivotal phase in preparing the samples for high-throughput sequencing, utilized the NEBNext^® Ultra™ RNA Library Prep Kit from Illumina^® (NEB, Ipswich, MA, USA). This was followed by sequencing on an Illumina NovaSeq 6000 platform, facilitated by Novogene Co., Ltd. (Beijing, China), to generate a broad and detailed transcriptomic dataset.

Differentially expressed genes (DEGs) were analyzed among the various sample groups using the DESeq2 R package version 3.0.3 [38]. DEGs were identified based on fold change (FC) ≥ 0 criteria and a p value < 0.05, ensuring statistical rigor in detecting significant transcriptional variations. Furthermore, Weighted Gene Co-expression Network Analysis (WGCNA) was applied to these DEGs using the WGCNA R package (version 3.5.0). This analysis facilitated the identification of co-expression gene networks, providing insights into the regulatory mechanisms and potential biological pathways influenced by N availability at this critical growth stage.

2.4. Metabolomic Analysis

During the 2021 season, metabolomic analyses were conducted on the fourth leaf and root samples collected 45 DAP, employing the methodologies described by Want et al. [39] and Zhang et al. [40] for metabolite extraction and analysis. These metabolites were subsequently annotated utilizing databases such as the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Human Metabolome Database (HMDB), and Lipidmaps to ensure comprehensive identification and classification.

For the analysis of the metabolomic data, both Principal Component Analysis (PCA) and Partial Least Squares-Discriminant Analysis (PLS-DA) were performed using the meta software package (R version 3.5.1) [41]. This software is recognized for its flexibility and comprehensive capabilities in processing metabolomics data, facilitating robust statistical analyses. Metabolites were identified as differentially accumulated based on specific criteria: a Variable Importance in Projection (VIP) score greater than 1, a p-value less than 0.05, and a fold change (FC) exceeding 1.2 or falling below 0.833. This stringent selection process ensured that only metabolites with significant differences in concentration between treatment groups were considered for further analysis.

2.5. Quantitative Real-Time PCR (qRT-PCR)

To quantify gene expression in potato leaves and roots, total RNA was extracted utilizing the Tiangen RNA Pure Plant Kit (Tiangen, Beijing, China). First-strand cDNA was synthesized employing the Hifair^® III 1st Strand cDNA Synthesis SuperMix (Yeasen, Shanghai, China) for qPCR (including gDNA Digester Plus) kit, which enables the preparation of template cDNA for subsequent PCR reactions.

Quantitative real-time PCR was performed in a reaction volume of 20 µL using the Hieff^® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China), chosen for its sensitivity and specificity for amplifying target sequences. The U6 gene was selected as the reference for normalization of gene expression data due to its established stability and reliability across various experimental conditions [42]. Gene-specific primers were meticulously designed with the PrimerQuest Tool (available at http://sg.idtdna.com/Primerquest/Home/Index (accessed on 8 January 2024)), ensuring optimal annealing and amplification efficiency for each target gene.

Relative quantification of gene expression levels was achieved through the 2^−ΔΔCT method, a robust and widely accepted approach for analyzing qRT-PCR data, providing insights into the relative changes in gene expression across different experimental conditions [43,44]. The sequences of the primers utilized in this assay are detailed in Supplementary Table S3, providing a comprehensive overview of the molecular tools applied in this study.

2.6. Statistical Analysis

Statistical evaluations of the data were meticulously performed using one-way Analysis of Variance (ANOVA) followed by Duncan’s Multiple Range Test for post-hoc analysis, setting the significance threshold at p < 0.05. These analyses were conducted utilizing the SPSS 22.0 Statistics software package (IBM Inc., Chicago, IL, USA), renowned for its robust statistical capabilities.

For the graphical representation of our findings, GraphPad Prism 8 (GraphPad, Boston, MA, USA) was employed. This software facilitated the creation of visually appealing and informative graphs that succinctly convey the experimental results.

Further, advanced bioinformatics analyses, including cluster heatmaps, PCA, and PLS-DA, were executed on the Metware Cloud platform (https://cloud.metware.cn (accessed on 26 May 2024)). This online resource offers a comprehensive suite of tools for the in-depth analysis of complex datasets, allowing for the elucidation of patterns and relationships within the data.

3. Results

3.1. Evaluation of Nitrogen Efficiency-Related Indicators in Two Potato Varieties

As N application rates increased, the tuber yields of both potato varieties exhibited a corresponding enhancement. In 2019, compared to the no N application, the yields of NS7 under LN and NN conditions increased by 8.54% and 26.58%, respectively. In contrast, XS6 demonstrated yield increases of 17.98% and 40.02% under the same conditions. A similar trend was observed in 2020, where NS7 yields increased by 11.18% and 31.49% under LN and NN conditions, respectively, while XS6 exhibited increases of 28.11% and 64.86%, respectively (Figure S2A). These results indicate that XS6 possesses a significantly higher yield potential compared to NS7, with this trend remaining consistent across both years. Furthermore, the apparent nitrogen use efficiency, nitrogen agronomic efficiency, and nitrogen contribution rate all improved with increasing N application, with XS6 consistently outperforming NS7 across these metrics (Figure S2B,C,F). Under LN conditions, compared to NN levels, the biological efficiency of NS7 and XS6 increased by 6.48% and 10.07%, respectively, in 2019, and by 6.72% and 11.36% in 2020, respectively (Figure S2D). Although nitrogen partial productivity decreased as N application rates increased, XS6 consistently exhibited superior performance relative to NS7 across all N levels (Figure S2E). Overall, XS6 demonstrated higher efficiency and greater yield potential in N utilization, highlighting its significant advantage in N management practices.

3.2. Physiological Responses of Two Potato Varieties to Nitrogen Supply

Our study assessed the impact of N supply on the growth of two potato cultivars, XS6 and NS7, by examining a range of physiological parameters. These included chlorophyll a, chlorophyll b, total chlorophyll content, and N content during the tuber expansion stage (45 DAP), and tuber yield and NUE at the tuber maturity stage (85 DAP). The experiments were structured around three nitrogen application scenarios: no nitrogen (0 kg·hm⁻²), LN (150 kg·hm⁻²), and NN (300 kg·hm⁻²).

Comparative analysis revealed that XS6 consistently outperformed NS7 under normal and LN conditions, demonstrating notably superior growth performance. Specifically, under LN conditions, XS6′s chlorophyll a, chlorophyll b, total chlorophyll, and N content were significantly higher than those of NS7, showing increases of 7.89%, 13.44%, 9.25%, and 13.35%, respectively. This was paralleled by a significant enhancement in tuber yield for XS6 across all N levels compared to NS7. Furthermore, XS6 exhibited a higher NUE than NS7, which was particularly noticeable at the tuber swelling stage under LN conditions, indicating a more efficient agronomic NUE for XS6.

These findings underscore the N-efficient characteristics of XS6 compared to the N-inefficient NS7 across the examined physiological parameters (Figure 1).

Additionally, our examination of the four key enzymes nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) revealed their activities increased alongside rising N supply for both cultivars. Remarkably, XS6 demonstrated significantly enhanced NR, GS, and GOGAT activity levels in both leaves and roots compared to NS7 under varying N conditions. Although no notable differences in GDH activity were observed under nitrogen fertilization, a significant increase in activity was recorded for XS6 under both LN and NN conditions (Figure 2).

3.3. Transcriptomic Analysis

3.3.1. Sequencing and Identification of Differentially Expressed Genes (DEGs)

In our quest to elucidate the genetic underpinnings of NUE in potatoes, we embarked on a comprehensive high-throughput transcriptomic analysis employing RNA sequencing (RNA-seq). This analysis encompassed 24 samples, each represented by three biological replicates from both XS6 and NS7 cultivars, under sufficient N and N starvation conditions. Utilizing the Illumina sequencing platform, we generated a substantial volume of data, comprising 156.67 Gb of raw reads, which were refined to 148.95 Gb of clean reads. These meticulously curated transcriptomic data have been deposited in the NCBI’s Sequence Read Archive (SRA) under accession number PRJNA1077475. A quality assessment of this dataset highlighted its excellence, with over 97.41% of bases achieving a Q20 score and the Q30 base percentage surpassing 92.83%. The GC content stood at 42.24%, underscoring the high fidelity of our sequencing efforts (Table S4). A distinct disparity was observed in the mapping rates to the potato reference genome between the two cultivars, with XS6 demonstrating a significantly higher alignment efficiency than NS7. This discrepancy suggests inherent genetic differences between the N-efficient XS6 and the N-inefficient NS7, laying the groundwork for further bioinformatic analyses.

PCA further delineated the variances within and between groups, attributing 27.69% of the leaf variation and 31.99% of the root variation to the primary principal component (PC1). This analysis facilitated the distinction of samples based on cultivar (PC1) and N treatment (PC2), reinforcing the reliability of our data as evidenced by correlation coefficients exceeding 0.95 among replicates (Figure 3A–C).

Our differential expression analysis, structured around four conditions (XS6_L(LN/NN), XS6_R(LN/NN), NS7_L(LN/NN), and NS7_R(LN/NN)), unveiled 2648 DEGs in NS7 leaves and 2984 in its roots, contrasting with 2372 DEGs in XS6 leaves and 3703 in roots when modulating N supply. Noteworthily, XS6 surpassed NS7 in the tally of DEGs across both tissues, indicating heightened adaptability to N-deficient stress (Figure 3D–F).

3.3.2. Enrichment Analysis of DEGs: GO and KEGG Pathways

We employed GO and KEGG analyses to elucidate the functions and involvement of DEGs in biological pathways, as illustrated in Figure S3 and Figure 4. Among the 45 significantly enriched GO terms, a substantial number were linked to biological processes and molecular functions. Specifically, in the NS7 leaves, there was notable enrichment for enzyme inhibitor activity (GO:0004857), enzyme regulator activity (GO:0030234), and DNA-binding activity (GO:0003700). Contrasting with this, the roots primarily showed enrichment for antioxidant (GO:0016209) and peroxidase activities (GO:0004601).

Intriguingly, both leaves and roots of the XS6 cultivar demonstrated significant enrichment for DNA-binding transcription factor activity (GO:0003700) and transcription regulator activity (GO:0140110) under LN conditions, suggesting a robust genetic response to N stress (Figure S3).

Subsequent analysis through the KEGG pathway database revealed enrichment in several key pathways across all comparison groups, including plant hormone signal transduction (sot04075) and phenylpropanoid biosynthesis (sot00940). Notably, the pathways related to the processing in the endoplasmic reticulum (sot04141) were significantly enriched in both NS7 and XS6 leaves. In contrast, pathways such as the citrate cycle (sot00020), carbon metabolism (sot01200), and N metabolism (sot00910) showed significant variations in NS7 roots, with XS6 roots displaying fewer changes. Moreover, enrichment was observed in pathways related to both general and specialized metabolism, such as sulfur metabolism (sot00920) and photosynthesis–antenna proteins (sot00196), along with the biosynthesis of amino acids, flavonoids, stilbenoids, diarylheptanoids, and gingerol.

These findings highlight the unique molecular responses of the two potato cultivars under LN stress, shedding light on the complex regulatory mechanisms at play. Figure S3 and Figure 4 visually represent the significantly enriched pathways identified through our GO and KEGG pathway analyses, respectively, underscoring the potential impact of these genetic pathways on the potato’s response to N availability.

3.4. Weighted Gene Co-Expression Network Analysis (WGCNA) of DEGs

Our analysis further delved into the complexities of N metabolism in XS6 and NS7 potato cultivars through Weighted Gene Co-expression Network Analysis (WGCNA), focusing on enzymes critical to N metabolism enzymes—nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT). The analysis yielded 18 distinct gene modules (Figure 5A,B), among which the blue module demonstrated a significant positive correlation with NR, GS, and GDH activities but a negative correlation with GOGAT activity. Conversely, the turquoise module exhibited inverse correlation patterns (Figure 5A). Notably, genes within the blue module predominantly showed increased expression under LN stress, with XS6 surpassing NS7 in expression levels within leaves (Figure S4). This pattern suggests a pivotal role of the blue module’s genes in N assimilation and utilization, prompting further examination.

Gene Ontology (GO) analysis of the blue module genes highlighted substantial enrichment in the Biological Process (BP) terms associated with cellular lipid metabolism, small molecule metabolism, and alpha-amino acid metabolism. The Cellular Component (CC) terms were predominantly linked to photosynthetic systems, while the Molecular Function (MF) terms included activities related to phosphatases, isomerases, gated channels, and N-acetyltransferases (Figure S5A, Table S5).

KEGG pathway enrichment analysis underscored the prominence of pathways integral to carbon and N metabolism, such as carbon metabolism, photosynthesis–antenna proteins, carbon fixation, carotenoid biosynthesis, amino acid biosynthesis, and starch and sucrose metabolism (Figure S5B). The KEGG classification revealed several genes in plant hormone signal transduction and metabolic pathways vital for responding to LN stress. This included genes for serine/threonine-protein kinase SRK2I, auxin-induced protein 15A, and ARR5, alongside a gene for beta-amylase involved in starch and sucrose metabolism, and genes within the TCA cycle like malate dehydrogenase and isocitrate dehydrogenase. The high-affinity nitrate transporter 2.7 and ferredoxin-nitrite reductase NIR were particularly interesting in the N metabolism pathway, underscoring their roles in N transport and assimilation (Figure S5C).

This comprehensive analysis through WGCNA not only elucidates the genetic basis of N utilization in potato cultivars but also pinpoints specific biological processes and pathways that are potentially critical for enhancing NUE under LN conditions.

3.5. Metabolomic Profiling under LN Stress

3.5.1. Metabolomic Data Quality Control

In this investigation, we applied non-targeted metabolomics using LC-MS/MS techniques to explore the spectrum of plant metabolites influenced by LN nutrition in potato roots and leaves. This advanced approach aimed to delineate the adaptive responses of the potato plant’s root and leaf systems to LN conditions. Our extensive analysis successfully identified a total of 1252 metabolites, which were systematically classified into 10 distinct categories including phenylpropanoids and polyketides, organoheterocyclic compounds, organic oxygen compounds, and several others, as shown in Figure 6A. Notably, lipids, organic acids, and phenylpropanoids emerged as the most prevalent classes among the identified metabolites, underscoring their potential role in the plant’s response to LN stress (Table S6).

PCA was conducted to discern the primary factors influencing metabolomic variability in leaves and roots. In leaf samples, PC1 and PC2 accounted for 37.23% and 15.53% of the total variation, respectively, with PC1 distinguishing samples by variety and PC2 by N treatment. A similar pattern was observed in root samples, where PC1 and PC2 explained 31.89% and 15.17% of the variation, respectively. This analysis revealed that the potato varieties predominantly drive metabolite variations, whereas N treatment is a secondary determinant (Figure 6B,C).

3.5.2. Identification and Analysis of Differentially Accumulated Metabolites (DAMs)

Our analysis utilized PLS-DA to pinpoint metabolites whose accumulation responded significantly to N application. The model evaluation parameters Q² > 0.5, R² X and R² Y are close to 1, underscoring the model’s robustness and reliability (Figure S6). For the identification of DAMs, we integrated VIP values with fold change (FC) criteria, selecting metabolites with VIP > 1.0 and significant fold changes (FC > 1.2 or FC < 0.833, p < 0.05) as differentially accumulated. This approach led to the identification of 146 DAMs in XS6 (29 upregulated, 117 downregulated) across leaves and roots under LN vs. NN conditions and 86 DAMs (30 upregulated, 56 downregulated) specifically in roots (Figure 7A). Conversely, NS7 exhibited 155 DAMs (106 upregulated, 49 downregulated) in leaves and 125 DAMs (80 upregulated, 45 downregulated) in roots, revealing higher variability in DAMs compared to XS6, aligning with our transcriptomic findings.

Venn diagrams facilitated the distinction of cultivar-specific DAMs and those shared between XS6 and NS7 under varying N conditions (Figure 7B,C). There were 105 DAMs in leaves and 73 in roots that were unique to XS6, while NS7 presented 114 unique DAMs in leaves and 112 in roots. Shared DAMs amounted to 41 in leaves and 13 in roots, indicating a common metabolic response to LN stress alongside cultivar-specific adaptations.

Heatmap cluster analyses further elucidated the expression patterns of shared DAMs, delineating three distinct groups in both leaves (Figure 7D) and roots (Figure 7E). In leaves, Group I’s 21 metabolites predominantly showed decreased accumulation under LN stress across both cultivars, featuring organoheterocyclic compounds and organic acids. Group II comprised 10 metabolites with increased accumulation in NS7 but decreased presence in XS6 under LN stress, and Group III consisted of 10 metabolites with increased accumulation in both cultivars under LN stress. The root analysis mirrored these findings, with Group I showing decreased, Group II showing mixed, and Group III showing increased metabolite accumulation under LN stress across both cultivars.

3.5.3. Pathway Enrichment Analysis of Differentially Accumulated Metabolites

KEGG pathway enrichment analysis was utilized to elucidate the metabolic pathways implicated in the response of potato roots and leaves to low N conditions. This analysis identified 62 key metabolic pathways associated with metabolite expression under varying N concentrations. To facilitate a direct comparison of metabolic pathway differences between groups, the top 20 pathways exhibiting the highest level of enrichment were selected for each comparison group through enrichment and topological analysis (Figure S7).

In the leaf comparison between XS6 and NS7, several pathways were distinctly enriched including ‘tropane, piperidine, and pyridine alkaloid biosynthesis’, ‘vitamin B6 metabolism’, ‘zeatin biosynthesis’, and ‘glycine, serine, and threonine metabolism’. Specifically, XS6 leaves demonstrated significant enrichment in pathways related to ‘photosynthesis’, ‘nicotinate and nicotinamide metabolism’, and ‘glutathione metabolism’. Conversely, the ‘starch and sucrose metabolism’ and ‘galactose metabolism’ pathways were predominantly enriched in NS7 leaves.

Root comparisons between XS6 and NS7 similarly highlighted enriched pathways such as ‘arginine and proline metabolism’, ‘beta-alanine metabolism’, and ‘phenylpropanoid biosynthesis’. XS6 roots were characterized by significant pathway enrichments in ‘photosynthesis pantothenate and CoA biosynthesis’, ‘purine metabolism’, and ‘nicotinate and nicotinamide metabolism’. NS7 roots, on the other hand, showed notable enrichment in ‘pyrimidine metabolism’.

3.6. Joint Transcriptomic and Metabolomic Pathway Analysis

Our comprehensive analysis sought to integrate the differential expression profiles from transcriptomic and metabolomic data, contrasting LN conditions against NN conditions. This integrative approach enabled the enrichment of identified DEGs and DAMs within KEGG pathways, offering insights into the metabolic and regulatory networks involved in N stress responses (Figure S8).

In the leaves of XS6, 36 KEGG pathways were discerned from the combined omics datasets, compared to 27 pathways in NS7 leaves. Among these, 18 pathways were common to both cultivars, indicating shared metabolic responses to LN stress. However, each cultivar also exhibited unique pathway enrichments: XS6 leaves showed exclusivity in pathways such as the citrate cycle (TCA cycle), photosynthesis, and alanine, aspartate, and glutamate metabolism, suggesting a distinct adaptive strategy to LN stress. Conversely, NS7 leaves uniquely enriched pathways, including biosynthesis of amino acids, ABC transporters, and starch and sucrose metabolism, underscoring differences in metabolic prioritization under N limitation (Figure 8A).

Root analyses mirrored this pattern, with XS6 and NS7 annotating 28 and 15 KEGG pathways, respectively. Shared pathways between the cultivars largely pertained to the biosynthesis and metabolism of compounds such as phenylpropanoid, arginine, proline, and glutathione, which are essential for stress responses. XS6 roots uniquely enriched 18 pathways related to phenylalanine metabolism, purine metabolism, and pyruvate metabolism, highlighting a comprehensive metabolic adaptation to LN stress (Figure 8B).

3.7. Examination of Key Metabolic Pathways under LN Stress

Our integrated analysis of the transcriptome and metabolome data sheds light on the significant impact of N availability on carbon and N compound contents in potato plants. Specifically, critical processes, including N absorption and assimilation, starch and sucrose metabolism, glycolysis, and the TCA cycle, were found to differ markedly under LN and NN conditions (Figure 9).

In XS6, the sucrose, trehalose, and maltose levels were observed to be lower than those in NS7, suggesting XS6′s higher sucrose utilization efficiency under LN conditions. This potentially facilitates an enhanced energy supply, with the transcriptional analysis revealing an upregulation trend for most genes involved in this metabolic pathway in leaves of both cultivars, more pronounced in XS6. Conversely, roots predominantly displayed a downregulation trend, indicating a strategic shift to energy production in leaves under LN stress.

The glycolysis pathway, initiating with sucrose conversion by hexokinase (HK) to fructose-6-P and culminating in the production of pyruvic acid from phosphoenolpyruvate by pyruvate kinase (PK), saw increased expression of HK and PK genes in XS6 leaves compared to NS7 under LN conditions. This suggests enhanced glycolysis activity in XS6, indicating a more intense metabolic response to LN stress. Pyruvate, produced via glycolysis, enters mitochondria for conversion to acetyl-CoA, feeding into the TCA cycle. The upregulation of pyruvate dehydrogenase (PDH) supports this metabolic flux, with XS6 showing superior expression levels over NS7.

Within the TCA cycle, the observation of decreased fumaric acid levels under LN treatment, coupled with the downregulation of key enzymes such as citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase, hints at diminished TCA cycle intensity under LN conditions.

Nitrogen metabolism analysis revealed the superior capacity of XS6 over NS7 in N transport, assimilation, and utilization, as evidenced by the higher expression of nitrate transporter (NRT), nitrate reductase (NR), and nitrite reductase (NiR) genes. The ammonium transporter gene (AMT) was downregulated in both cultivars under LN conditions, albeit to a lesser extent in NS7, while the expression of glutamine synthetase (GS) gene was notably upregulated in XS6 leaves, underscoring its efficiency in N management.

3.8. Validation of RNA-Seq Results through qRT-PCR

To validate the expression patterns of pivotal candidate genes identified through RNA sequencing, quantitative real-time PCR (qRT-PCR) was conducted. This validation process is crucial for confirming the reliability of RNA-seq findings (Figure 10). The comparative analysis of gene expression levels, as determined by qRT-PCR, displayed a significant concordance with the RNA-seq data. This high degree of correlation, quantified by a correlation coefficient (R²) of 0.86, underscores the consistency between these two methodologies in measuring gene expression levels (Figure S9).

4. Discussion

The pressing challenge of N scarcity significantly hampers global agricultural productivity, highlighting the urgent need for crop varieties with heightened NUE. Current agricultural practices, marked by excessive N fertilizers, face issues such as low N utilization rates and substantial environmental pollution. Addressing these challenges necessitates a deeper understanding of the complex regulatory mechanisms that govern NUE. Although previous research has explored the effects of N deficiency on the expression of N-responsive genes [45,46], the molecular distinctions between varieties differing in NUE have been less understood. This study utilized two potato varieties XS6 and NS7, with contrasting NUE, to explore the molecular mechanisms underlying NUE in response to LN and NN application conditions. Our investigation revealed significant physiological differences between the two varieties, likely influenced by gene expression and metabolite level variances. This discussion will center on these physiological distinctions and highlight the genes and pathways crucial for enhancing carbon and N metabolism in potato genotypes under LN stress conditions, aiming to improve NUE. Integrating transcriptomic and metabolomic data, our study underscores significant modifications in metabolic processes such as N absorption, assimilation, and sucrose metabolism under varying N conditions. This suggests that XS6 may exhibit higher sucrose utilization efficiency under LN conditions, thus ensuring an adequate energy supply for growth. This integrative approach provides a comprehensive understanding of the physiological traits and molecular pathways that contribute to the observed differences in NUE between XS6 and NS7, offering valuable insights for developing strategies to combat N limitation and enhance NUE in crop production.

4.1. Physiological Characteristics That Contribute to High NUE in Potato

Recent research underscores that enhancements in Nitrogen Uptake Efficiency (NupE) and Nitrogen Utilization Efficiency (NutE) are essential for significant improvements in crop NUE [5,13,47]. Identifying the physiological and molecular mechanisms contributing to NUE emerges as a pivotal strategy for its improvement. LN stress adversely affects plant growth and physiological traits, leading to diminished crop yield and quality—a phenomenon consistent across various crops, including Brassica napus [6], maize [22], wheat [40], apple [48], and rice [49]. Our field experiment on potato, an N-intensive crop, involved two genotypes to assess NUE variations. The N-efficient genotype XS6 consistently demonstrated higher chlorophyll content, N levels, and tuber yield under LN conditions compared to the N-inefficient genotype NS7. This suggests a superior capability of XS6 in N uptake and carbon assimilation, irrespective of N supply levels.

Nitrogen assimilation, a critical process facilitated by enzymes such as NR, GS, GOGAT, and GDH, plays a significant role in this context [50]. The efficient conversion of ammonium, whether absorbed directly or derived from nitrate reduction, into vital amino acids via the GS/GOGAT cycle indicates the plant’s N assimilation efficiency [14]. Under LN stress, XS6 displayed enhanced activity of these enzymes compared to NS7, implying a robust capacity for amino acid biosynthesis under N-deficient conditions. This enhanced N assimilation in XS6 potentially underpins the observed improvements in NUE. Furthermore, it is noted that amino acid concentrations in the phloem sap are significantly higher than in the cytoplasm of mesophyll cells, suggesting efficient loading and transport mechanisms [51]. However, the detailed mechanisms behind amino acid transport in XS6 and NS7 require further investigation to elucidate their contributions to the genotypes’ differential NUE.

4.2. Molecular Mechanism and Regulation of Carbon and Nitrogen Metabolism

C and N metabolism are intricately interconnected, playing pivotal roles in the biological systems of various organisms [52,53]. Carbon assimilation, through processes such as carbon fixation, supplies the necessary carbon skeletons for nitrate (${\mathrm{NO}}_3^{-}$) assimilation, highlighting the integral relationship between these metabolic pathways [54]. Our study delved into the gene expression dynamics within key metabolic pathways, including starch and sucrose metabolism, glycolysis/gluconeogenesis, the TCA cycle, and N metabolism. This exploration revealed distinct gene expression patterns, underscoring the complex regulatory mechanisms orchestrating carbon and nitrogen metabolism in response to environmental and physiological cues.

Carbohydrates such as sucrose and starch are crucial for plant development, serving as key structural elements, energy reservoirs, and osmotic regulators [55]. Notably, N deficiency stress often leads to an augmented accumulation of these carbohydrates, a response aimed at compensating for the impaired N metabolism [56]. In line with previous studies, our investigation observed an increased sucrose concentration in the leaves of both XS6 and NS7 potato varieties under N deficiency. This elevation in sucrose content may be largely attributed to the enhanced expression of sucrose phosphate synthetase (SPS) and sucrose phosphatase (SPP), enzymes that are pivotal for sucrose biosynthesis in plant foliage. Moreover, our analysis revealed an upregulation in genes involved in trehalose metabolism, particularly TPS and TP, across both XS6 and NS7 leaves under LN conditions. This upregulation likely contributes to trehalose synthesis, bolstering the plants’ adaptive response to LN availability. Trehalose, recognized for its critical role as a stress regulator and signaling molecule, has been documented to mitigate adverse effects of drought and salt stress in plants, further underscoring its significance in stress resilience and adaptation mechanisms. Our findings align with the broader body of research highlighting the adaptive strategies plants employ to navigate N deficiency stress, focusing on the modulation of carbohydrate metabolism pathways [57,58].

Glycolysis and the TCA cycle are cornerstone metabolic pathways intricately linked to each other, serving as fundamental conduits for energy production in living organisms [59]. Glycolysis, in particular, has been identified as a crucial energy-generating mechanism, with hexokinase (HK) and pyruvate kinase (PK) recognized as key rate-limiting enzymes within this pathway [60]. Our study observed a significant upregulation of HK and PK genes in potato leaves, especially pronounced in the XS6 variety compared to NS7. This upregulation underscores their potential role in enhancing glycolysis rates under N-deficient conditions, bolstering the potatoes’ adaptability to such stress.

Simultaneously, the TCA cycle, a pivotal energy source for cellular metabolism [61], showed an overall upregulation of related metabolites and genes in both XS6 and NS7 varieties under LN and control conditions, albeit with certain exceptions like downregulation of the IDH gene [62,63]. This pattern of gene expression, which includes downregulation of key enzymes such as citrate synthase (CS), aconitase (ACO), and fumarase (FUM) in XS6 leaves, aligns with observations in other plant species, indicating a nuanced response to N stress.

In higher plants, N assimilation predominantly occurs through the GS/GOGAT cycle [64], transforming inorganic N in soils into usable forms within the plant. This study highlights the increased expression of specific NRT genes in XS6, suggesting an enhanced capacity for nitrate transport compared to NS7 [65]. Moreover, genes involved in N reduction and assimilation, including NR and NiR, were upregulated in leaves of both varieties but downregulated in roots, indicating differential regulation based on tissue type. The exclusive upregulation of a GS gene and GOGAT genes in XS6 leaves suggests a variety-specific response to N assimilation under stress conditions [8].

These findings underscore the complexity of the molecular mechanisms regulating carbon and N metabolism in response to N availability, offering insights into potential genetic targets for enhancing NUE in crops. By manipulating genes associated with N assimilation, as seen in various crops through upregulation of genes like TaGS2-2 in wheat [66], HvGS1-1 in barley [67], and Gln1-3 in maize [68], it is possible to significantly improve NUE and crop yield [69]. Similarly, the upregulation of the OsGOGAT 1 gene in rice underscores the role of enhanced N absorption and assimilation in increasing NUE and yield, providing a promising avenue for agricultural improvement.

4.3. Hypothesis

In this study, we have conceptualized a model delineating the potential molecular mechanisms that underpin the enhanced NUE in potatoes under both LN and NN conditions. Our comparative analysis between the XS6 and NS7 potato varieties highlights three pivotal characteristics contributing to the superior NUE observed in XS6:

(1)

Enhanced photosynthesis: XS6 exhibits a higher chlorophyll content than NS7, laying a robust foundation for improved photosynthesis and increased organic matter production.

(2)

Efficient nitrogen uptake and accumulation: During the critical tuber expansion stage, XS6 demonstrates remarkable N uptake and accumulation capability. This efficiency is underpinned by the sustained high expression levels of NRT and nitrate reductase NR genes, even under N-deficient conditions. Such molecular adaptation facilitates greater N uptake from the soil and its accumulation in the plant’s aboveground tissues, effectively reducing N leakage into the groundwater.

(3)

Role of trehalose in alleviating nitrogen deficiency: The observed upregulation of trehalose in both XS6 and NS7 under LN conditions suggests a crucial role for this sugar in mitigating the effects of N deficiency. Furthermore, N stored in aboveground tissues is potentially mobilized and redirected to the roots and tubers, supporting sustained development despite periods of N scarcity.

Our model provides a comprehensive overview of the molecular dynamics governing N absorption and assimilation in potatoes, offering valuable insights into the mechanisms enhancing NUE. This understanding is pivotal for developing strategies to improve NUE in crop plants, thereby addressing one of the critical challenges in contemporary agriculture (Figure 11).

5. Conclusions

Comprehensive physiological, transcriptomic, and metabolomic analyses were performed on two potato cultivars subjected to LN stress conditions, unveiling significant variations between the XS6 and NS7 cultivars. The XS6 cultivar demonstrated notable enhancements with statistical significance (p < 0.01) in key physiological metrics such as chlorophyll content, N content, NUE, tuber yield, and activity of N assimilation enzymes in contrast to NS7. The transcriptomic investigation further revealed a greater abundance of DEGs related to C and N metabolism in both the leaves and roots of XS6, suggesting a robust genetic response to LN stress in this N-efficient potato variety. Consequently, a conceptual model was developed to elucidate the complex interplay of C and N metabolism that underpins the observed high NUE, suggesting that the coordinated efficiency of N absorption and assimilation by the roots, alongside enhanced energy production in the shoots and leaves, significantly contributes to elevated NUE. Additionally, the metabolomic analysis highlighted trehalose’s beneficial impact in alleviating N deficiency and augmenting NUE. These findings offer profound insights into adaptive strategies for overcoming LN stress and enhancing NUE in potato cultivation, presenting promising avenues for future agricultural innovation and sustainability.