2.1 Sequencing data analysis and validation
Analysis of the quantity and quality of the transcriptome sequencing data showed that there were 4 × 107 clean reads, corresponding to 6 Gb of sequencing data per sample. The Q30 ratio was ≥ 94% for each treatment group. These results indicated that the sequencing data from all samples were of high quality and met the standards for use in subsequent analyses (Supplementary Table 2). To verify the transcriptomic data, 20 differentially expressed genes (DEGs) were randomly selected from different treatment groups for validation via RT-qPCR. The RT-qPCR results were consistent with the transcriptomic data (R2 = 0.8235), indicating that the sequencing results were reliable (Supplementary Fig. S1).
2.2 Tissue-specific variation in water- and nitrogen-responsive genes
Gene expression was analyzed in potato root and leaf tissues using pairwise comparisons of treatment groups, namely W1N3 vs. W3N3; W1N1 vs. W3N1; W3N1 vs. W3N3; and W1N1 vs. W1N3. A total of eight sets of DEGs were identified from the four treatment group comparisons in two tissues (Fig. 1a). The W1N1 vs. W1N3 comparison showed the largest number of DEGs. In both leaves and roots, there were more DEGs between treatments with variation in nitrogen levels than between treatments with variation in water levels (Fig. 1b). A crossover analysis of DEGs revealed that there were 14 DEGs in leaves that responded to changes in both water and nitrogen (Fig. 1c, Supplementary Table 3); in roots, that number increased to 19 DEGs (Fig. 1d, Supplementary Table 4). In potato leaves and roots, there were 693 and 1224 DEGs, respectively, between nitrogen treatments, and 287 and 438 DEGs, respectively, between water treatments. These data indicated that there were more DEGs in response to changes in nitrogen levels than water levels, and that there was an interaction between responses to water and nitrogen. Plants require functionally related genes to regulate and coordinate stress adaptation and growth; TFs in the DEG sets were therefore analyzed. These TFs were found to have a mutual regulatory relationship. The connectivity was higher for TF regulatory networks in potato roots than in leaves, although there was some crosstalk between the regulatory networks in the two tissues. MIKC_MADS (PGSC0003DMT400063312) was differentially expressed in both roots and leaves in response to increased nitrogen (W1N1 vs. W1N3), indicating that MIKC_MADS played an important role in nitrogen regulation in at least two tissues (Fig. 1e and f).
To further understand the functions of DEGs, GO enrichment analysis was performed (Supplementary Fig. S2). The results suggested that higher levels of water or nitrogen promoted root cell differentiation and proliferation; hormone and specialized metabolite biosynthesis; and accumulation of photosynthetic products in transit. In leaves, genes that were up-regulated in response to higher water or nitrogen levels were related to chlorophyll and specialized metabolite synthesis, photosynthetic product production and transport, and assimilation and transport of ions and organic matter from the soil. In roots, DEGs between nitrogen treatments were enriched in functions related to nitrogen absorption and assimilation, symbiotic fungi collaboration, miRNA-mediated gene silencing, and other biological processes. Genes that responded to changes in water levels in the roots were enriched in functions related to chromatin morphological changes, histone modifications, symbiotic fungal collaboration, and other biological processes.
2.3 Potato stress tolerance was increased under drought and nitrogen deficit
KEGG enrichment analysis was next conducted for DEGs between treatment groups. In response to increased nitrogen under drought conditions (W1N1 vs. W1N3), genes involved in glycolysis/gluconeogenesis and hormone signaling pathways were differentially expressed in roots, and genes related to photosynthesis, glycolysis/gluconeogenesis, amino acid metabolism, and the glutathione metabolism pathway were differentially expressed in leaves. In response to increased water under low nitrogen conditions (W1N1 vs. W3N1), genes related to specialized metabolites, signaling, and cellular processes were differentially expressed in roots, and genes related to glutathione metabolism, specialized metabolites, mitogen activated protein kinase (MAPK) signaling, and hormone signaling pathways were differentially expressed in leaves. In contrast, in response to increased nitrogen under adequate water conditions (W3N1 vs. W3N3) or increased water under adequate nitrogen conditions (W1N3 vs. W3N3), DEGs in both roots and leaves were primarily enriched in cell proliferation, gluconeogenesis, and other metabolic pathways related to cell growth and development (Supplementary Fig. S3). Plant height and root fresh weight were also significantly higher after increasing nitrogen levels under sufficient water conditions and after increasing water levels under sufficient nitrogen conditions. There were no significant changes in the fresh weight of either roots or leaves in response to variations in nitrogen or water levels while there was still a deficit in either input (Fig. 2).
Water-responsive genes in root tissue under sufficient nitrogen conditions were enriched in cell replication and proliferation, photosynthetic product metabolism, and photoassimilate accumulation; in leaf tissue, they were enriched in photosynthesis and amino acid metabolism pathways. In addition, root fresh weight and plant height increased in response to additional water under sufficient nitrogen conditions. DEGs in both root and leaf tissues were significantly enriched in pathways related to specialized metabolism and signal transduction. Moreover, plant height and fresh weight were lower under either water- or nitrogen-deficient conditions. This indicates that potato plants enhanced stress tolerance through up-regulation of specialized metabolism, signal transduction, and glutathione metabolism to adapt to low water or nitrogen conditions.
2.4 Changes in soil nitrogen or moisture levels led to induction of distinct metabolic pathways
2.4.1 Increased nitrogen application under drought conditions can improve the efficiency of light energy utilization in leaves
Analysis of genes related to photosynthetic metabolic pathways revealed that 79 genes involved in the light reactions were induced by increased nitrogen levels under drought conditions (W1N1 vs. W1N3). Many genes encoding components of the photopigment protein complex and the oxygen release complex were up-regulated. For example, the oxygen-evolving enhancer protein 2 − 1 gene (PGSC0003DMG400031395) had a log2FC value of 4.5 in this comparison; four genes encoding electron carriers were up-regulated, with one (PGSC0003DMG400004532) having a log2FC value of 4.2. Sixteen up-regulated genes were identified as components of the dark reactions, including two key rate-limiting fructose-1,6 − 2 phosphatase genes (PGSC0003DMG400019189 and PGSC0003DMG400024109) with log2FC values of 2.2 and 1.4, respectively. Some members of the Calvin–Benson–Bassham (CBB) cycle were also up-regulated, namely three glyceraldehyde 3-phosphate dehydrogenase genes and several ribulose-1,5-diphosphate carboxylase genes. Thirteen DEGs were associated with photorespiration, most of which were up-regulated (Supplementary Table 5). No significant changes in photosynthetic characteristics were identified when water levels were increased under limited nitrogen conditions. Measurements of physiological photosynthetic characteristics showed that the saturated vapor pressure difference and relative chlorophyll content increased in potato leaves in response to nitrogen addition under drought conditions, whereas the intercellular CO2 concentration, net photosynthetic rate, and stomatal conductance remained unchanged. The relative chlorophyll content and saturated water vapor pressure difference decreased and the net photosynthetic rate increased in potato leaves when water levels were increased under sufficient nitrogen conditions (Fig. 3).
The results discussed above suggest that increasing nitrogen levels under drought conditions can promote expression of genes encoding photopigment complex proteins, electron and proton transporter proteins, and oxygen release complex proteins, which in turn increase light energy capture, conversion efficiency, and water photolysis efficiency. Multiple rate-limiting enzyme-encoding genes in the CBB cycle were up-regulated, enhancing carbon assimilation efficiency. This also increased the saturated vapor pressure difference within leaf pulp cells and water utilization efficiency in leaves. Genes encoding a protein in the light-capturing complex and an electron transporter in the light reaction center were down-regulated, whereas an oxygen release complex gene was up-regulated. The relative chlorophyll content and saturated water pressure difference decreased, and the intercellular carbon dioxide concentration did not change. These results indicated that the limiting factor for photosynthesis under nitrogen sufficient conditions was water photolysis. The photoreaction centers in potato leaves actively decreased their capacity for light energy capture to prevent light damage caused by proton deficiency.
2.4.2 Nitrogen levels could affect expression of genes related to stress resistance
In response to changes in water or nitrogen levels, root and leaf cells showed differential expression of genes related to stress resistance, such as hormones (auxins, brassinolide, and ethylene), heat stress proteins, glutathione metabolism, signal transduction, redox reactions, specialized metabolism, and abiotic stress. In response to increased nitrogen levels under drought conditions (W1N1 vs. W1N3), there were 103 hormone-related and 182 cell wall construction-related DEGs in root tissues, such as genes encoding indoleacetic acid (IAA)-amino acid hydrolase 4, ethylene-responsive transcriptional coactivator, jasmonate o-methyltransferase, gibberellin 2-oxidase 1, glycosyl hydrolase family, glucan protein synthase, and β-1,3 glucan hydrolase. Many stress-related genes were also differentially expressed, including those encoding members of the heat shock protein family, oxidoreductase, and ubiquitination-related enzymes. In response to increased water under limited nitrogen conditions (W1N1 vs. W3N1), DEGs included 15 hormone-related genes, four cell wall construction-related genes, nine specialized metabolism-related genes, and several stress-responsive genes that were regulated in root tissues (Supplementary Table 6).
These results indicated that in addition to providing bioavailable nitrogen, urea can act as a signal that interacts with genes related to hormone pathways and specialized metabolism. Further analysis revealed that expression of genes related to ABA synthesis could only be detected in the root, not in the leaf. This suggested a unique regulatory role of roots in response to changes in water and nitrogen availability in the soil. ATHVA22C (PGSC0003DMG402023061), a member of the ABA pathway, was up-regulated in root tissue after nitrogen application under normal water or drought conditions (W1N1 vs. W1N3, log2FC = 2.17; W3N1 vs. W3N3, log2FC = 1.98), indicating that this gene had a key role in the interaction between nitrogen and ABA.
2.4.3 Nitrogen transporters exhibited tissue-specific expression in potato
Genes related to the nitrogen metabolism pathway were next identified and analyzed. Four high-affinity nitrate transporter genes were identified in root tissues. One of these (PGSC0003DMG400019674) was up-regulated in response to increased water or nitrogen (log2FC > 2.0), peaking at log2FC = 4.9 in response to increased nitrogen under standard water conditions (W3N1 vs. W3N3). Another high-affinity nitrate transporter (PGSC0003DMG401011998) and three ammonium transporter genes (PGSC0003DMG400046020, PGSC0003DMG401015894, and PGSC0003DMG400028710) were up-regulated in the roots in response to increased water but were down-regulated in response to increased nitrogen. The gene encoding ferredoxin-nitrite reductase (PGSC0003DMG400008262) was down-regulated in response to increased water and up-regulated in response to increased nitrogen.
The gene encoding glutamine synthetase cytosolic isozyme 1–1 (PGSC0003DMG400023620) was up-regulated in leaves in response to increased nitrogen but down-regulated in response to increased water levels. Three ammonium transporter genes were identified as DEGs in leaves, one of which (ammonium transporter 1 member 2 [PGSC0003DMG400028710]) was up-regulated in response to increased water levels and down-regulated in response to increased nitrogen (Supplementary Table 7). Tuber protein content was significantly higher under both adequate water and drought when nitrogen levels were increased (Supplementary Fig. S4).
These results showed that increased nitrogen fertilizer application promoted nitrogen uptake and transport via up-regulation of the high-affinity nitrate transporter and ferredoxin-nitrite reductase genes and down-regulation of the ammonium transporter gene in roots. In leaves, higher nitrogen levels led to up-regulation of the gene encoding glutamine synthetase cytosolic isozyme 1–1 to promote nitrogen assimilation and utilization. Together, these changes resulted in higher protein accumulation in tubers. High-affinity nitrate transporter 2.4-like genes were specifically expressed in roots, whereas ammonium transporter genes (PGSC0003DMG401015894 and PGSC0003DMG400028710) were expressed in both roots and leaves. These findings indicate that nitrogen uptake and utilization were mainly regulated by nitrate and ammonia transporters and that the nitrate transporter was specifically expressed in the root, whereas the ammonia transporter acted in both roots and leaves.
2.4.4 Increased nitrogen fertilization can delay potato tuber expansion
Potato tuber expansion is regulated by leaves. Genes such as StSP6A, StaGL8, and StPHYB2 are expressed in leaves, and the corresponding protein products can be transmitted to the stolon to cause stolon sub-apical expansion (Kondhare et al. 2021). Here, we measured expression of the genes involved in mediating tuber expansion in leaves. Significant differential expression of PATATIN, StSP6A, StPHYB2, and StaGL8 was observed. Specifically, PATATIN was up-regulated in response to increased nitrogen application and down-regulated in response to increased water levels. StSP6A and StaGL8 were down-regulated in response to increased nitrogen and up-regulated in response to increased water. Other regulatory genes were also found to be differentially expressed (Fig. 4a, Supplementary Table 8).
Further analysis of DEGs related to tuber expansion revealed that they could interact with each other. StSP6A, MADS BOX, and StE(z)2 were hub genes (Fig. 4b). Increased nitrogen levels led to down-regulation of StaGL8 and up-regulation of PATATIN and StPHYB2, eventually leading to down-regulation of StSP6A. Increased water levels led to down-regulation of PATATIN and up-regulation of StaGL8 and StPHYB2, which then led to up-regulation of StSP6A and promotion of stolon end extension. Clustering analysis of the gene expression profiles revealed that genes related to tuber expansion clustered together (i.e., showed similar expression patterns) in response to increased nitrogen, and that genes responding to increased water levels clustered into another group. Observations of potato stolon developmental morphology showed that changes in soil nitrogen content did affect stolon development. The stolon entered the tuber expansion stage in nitrogen-deficient conditions (W1N1 and W3N1), but was in the sub-apical expansion stage under sufficient nitrogen conditions (W1N3 and W3N3) (Fig. 4c). There was no difference in per-plant yield when nitrogen levels were increased under drought conditions, but there was a significant increase in yield when nitrogen levels were increased under sufficient water conditions or when water levels were increased (Fig. 4d). Thus, nitrogen limitation promoted tuber expansion and accelerated entry of stolons into the tuber expansion phase; however, this failed to increase yield. In contrast, high nitrogen treatment prolonged the period of stolon development. We hypothesize that expression of StaGL8, PATATIN, StPHYB2, and other regulatory genes in leaves in response to high nitrogen levels resulted in down-regulation of StSP6A, which inhibited stolon expansion and prolonged the vegetative growth period.
2.5 Screening of key genes responding to changes in water and nitrogen levels
A total of 32 modules were obtained from WGCNA (Fig. 5a). Tuber weight, plant height, and other indicators were significantly associated with these modules (Fig. 5b). The 14,458 genes in the turquoise module were significantly positively correlated with root tissue (r = 0.99; p = 2e-21). The 3,352 genes in the blue module were significantly positively correlated with leaf tissue and were specifically expressed under low nitrogen conditions (r = -0.83; p = 6e-07) (Fig. 5c and d). The blue module was significantly enriched in polysaccharide, amino acid, and specialized metabolite metabolic pathways; the turquoise module was significantly enriched in ubiquitination and protein degradation pathways (Supplementary Fig. S5). Protein interaction networks were drawn for the genes present in those two modules. Based on their expression levels (and a connectivity threshold of > 0.5), 34 candidate water- and nitrogen-responsive genes were selected (Supplementary Table 9, Fig. S6). Ten were selected at random for RT-PCR validation (Fig. 5e). The expression levels of these genes differed between the corresponding treatment and control groups, indicating that the 34 candidate genes played important roles in responding to changes in water and nitrogen levels.