Literature DB >> 33119630

Transcriptome analysis reveals Nitrogen deficiency induced alterations in leaf and root of three cultivars of potato (Solanum tuberosum L.).

Jingying Zhang1, Yaping Wang1, Yanfei Zhao1, Yun Zhang1, Jiayue Zhang1, Haoran Ma1, Yuzhu Han1.   

Abstract

n class="Chemical">Nitrogen (N) is a key element for the production of n class="Species">potato. The N uptake efficiency, N use efficiency and increased N utilization efficiency can be decreased by N deficiency treatment. We performed this study to investigate the association between transcriptomic profiles and the efficiencies of N in potato. Potato cultivars "Yanshu 4" (short for Y), "Xiabodi" (cv. Shepody, short for X) and "Chunshu 4" (short for C) were treated with sufficient N fertilizer and deficient N fertilizer. Then, the growth parameters and tuber yield were recorded; the contents of soluble sugar and protein were measured; and the activities of enzymes were detected. Leaf and root transcriptomes were analyzed and differentially expressed genes (DEGs) in response to N deficiency were identified. The results showed that N deficiency decreased the nitrate reductase (NR), glutamine synthetase (GS) and root activity. Most of the DEGs between N-treated and N-deficiency participate the processes of transport, nitrate transport, nitrogen compound transport and N metabolism in C and Y, not in X, indicating the cultivar-dependent response to N deficiency. DEGs like glutamate dehydrogenase (StGDH), glutamine synthetase (StGS) and carbonic anhydrase (StCA) play key roles in these processes mentioned above. DEGs related to N metabolism showed a close relationship with the N utilization efficiency (UTE), but not with N use efficiency (NUE). The Major Facilitator Superfamily (MFS) members, like nitrate transporter 2.4 (StNRT2.4), 2.5 (StNRT2.5) and 2.7 (StNRT2.7), were mainly enriched in the processes associated with response to stresses and defense, indicating that N deficiency induced stresses in all cultivars.

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Year:  2020        PMID： 33119630      PMCID： PMC7595393          DOI： 10.1371/journal.pone.0240662

Source DB:  PubMed          Journal:  PLoS One        ISSN： 1932-6203            Impact factor:   3.240


1. Introduction

n class="Species">Potato (n class="Species">Solanum tuberosum L.) is the most important non-grain food crop in the world, the 4th most important staple food crop and the 12th most important agricultural product [1]. Potato is rich in carbohydrates, vitamins, minerals and proteins that are essential for humans, its tuber is used as a staple food globally [1-3]. Nitrogen (N) is a necessary nutrient for crop growth, especially for the production of N-sensitive crop potato [1-4]. For potato pro duction, N content in soil is a limiting factor. Therefore, the utilization efficiency of N is extremely important for the growth of potatoes. N efficiency estimation has been widely used to measure the capacity of plants to acquire and utilize nutrients for biomass production [5]. The N use efficiency (NUE, calculated as the tuber yield per unit of nutrient supply from soil and fertilizer in potato) is an important index for the production and market value of potato. As an important agronomy practice, applying nitrogen fertilizer in potato cropping can greatly improve potato production [6]. Therefore, understanding the N responsiveness in potato is important for high-NUE potato varieties breeding. The ideal potato genotype has both high genetic NUE and high N reactivity [7,8]. Improving n class="Chemical">NUE is one of the most effective means to inpan>crease crop productivity while reducinpan>g environmental degradation and farmers’ costs [9]. n class="Chemical">NUE could be divided into N uptake efficiency (UPE), and N utilization efficiency (UTE, calculated as the tuber yield divided by the maximum plant N pool) [10]. The UTE represents the efficiency of assimilation and remobilization of plant N to ultimately produce grain [11]. Improving UTE can reduce N consumption, maintain (even increase) production and may reduce excessive input of nitrogen fertilizer. UTE ranges from 50% to 80% in potato, and may be affected by many factors, such as N fertilizer application, agronomic measures and plant genetic factors [9,12]. In order to use nitrogen more efficiently and economically, different approaches had been tried to reduce the use of N fertilizers while ensuring yield [13]. There are many agricultural practices in improving n class="Chemical">NUE and crop yield, inpan>cludinpan>g controllinpan>g plant density, sowinpan>g time, and breedinpan>g high-n class="Chemical">NUE plants [12,13]. For instance, Yin et al demonstrated that delaying sowing and reducing N fertilizer application could achieve comparable yields of wheat [9]. They reported that delaying sowing time could decrease the UPE and spike density, and increase the UTE and grain number, but not effects found on grain yields [9]. In addition to agronomic strategies, plant genetics information is also an important factor. There are various genes associated with N absorption and utilization, like nitrate transporters (NRTs), nitrate reductase (NR), glutamine synthetase (GS), glutamate dehydrogenase (GDH) and nitrite reductase (NiR) [12]. It has been reported that the GS overexpressing plant can increase the UTE and UPE in wheat and barley, which results in increasing crop yields [14,15]. Understanding the relationship of gene expression pattern and NUE is important for potato breeding. In the production practice, we found that the utilization efficiency of N of “Yanshu 4”, “Xiabodi” (cv. Shepody) and “Chunshu 4” n class="Species">potatoes are quite different inpan> the production practice. Accordinpan>g to production experience, unpan>der the condition of sufficient n class="Chemical">nitrogen fertilizer, “Yanshu 4” was a high-absorption but low-utilization potato, “Xiabodi” was a medium-absorption and medium-utilization potato, and “Chunshu 4” was a low-absorption but high-utilization potato. In this study, we performed this study to investigate the difference in NUE, UPE, UTE and gene expression profiles among these three potato cultivars. All these three potato cultivars were applied with N-complete and N-deficient fertilizer. The growth, production and physiology parameters were detected and analyzed in combination with transcriptome analysis. This study might add more information on N deficiency-induced molecular profiles.

2. Materials and methods

2.1. Plant and experiment design

Three n class="Species">potato cultivars “Yanshu 4” (Y for short), “Xiabodi” (cv. Shepody, X for short) and “Chunpan>shu 4” (C for short) were planted inpan>to pots inpan> an artificial climate chamber with conditions of 24°C, 60% humidity and 14:10 light/dark cycle. All n class="Species">potato tubers were obtained from the planting Resource Bank of Jilin Agricultural University. The potato plants of each cultivar were randomly divided into two groups: N-complete group (short for N) and N-deficient nutrient group (30 plants per group). Plants in N group were routinely treated with 3.3kg/100 m2 N (Urea), 1.8kg/100 m2 P (P2O5), and 2.7 kg/100 m2 K (K2O); Plants in N-deficient nutrient group were treated with 1.8kg/m2 P(P2O5) and 2.7kg/100m2 K (K2O), and no N fertilizer applied. The amount of fertilizer is converted according to the field production practice. Accordingly, samples were assigned into 6 groups according to the treatment and cultivar difference, including “Yanshu 4” treated with (short for YN) and without N (short for Y); “Xiabodi” treated with (short for XN) and without N (short for X); “Chunshu 4” treated with (short for CN) and without N (short for C). Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N notes treatment with 22 kg/667 m2 N. Each group was repeated for 4 times.

2.2. Measurement of physiological and biochemical parameters

Nine plants in each group were randomly selected, three of which were pooled as a repeat and a total of three replicates were set for each group. The fresh and dry weight (g/plant) of all the leaves in a plant were recorded at the mature stage. Also, the fresh and dry weight (g/plant) of stem and root of each plant were measured. Also, the total fresh tuber of each plant was weighted by a platform scale. The n class="Chemical">anthrone method [16] and n class="Chemical">Coomassie Brilliant Blue G-250 dye-binding method [17] were used to detect the contents of soluble sugars (carbohydrate) and soluble proteins of the flag leaf, respectively. The data of five developmental stages (seedling, bud, tuber bulking, starch accumulation and mature stages) were recorded continuously to evaluate the soluble sugars and soluble proteins variation. The activity of nitrate reductase (NR, EC 1.7.1.3) and glutamine synthetase (GS, EC-6.3.1.2) were detected using a glutamine synthetase assay kit (Nanjing Jiancheng, Bioengineering Institute, China) according to the manufacturer’s instructions. Briefly, the enzyme solution of plant tissues was extracted with sodium phosphate buffer. Then the color reaction was carried out and measured by a Microplate ELISA Reader (BioTek, USA). Then the enzyme activity was calculated based on the manufacturer’s instruction. Root activity directly affects the nutritional status and yield of the aboveground. Root activity was measured using triphenyl tetrazolium chloride (TTC) method reported previously [18]. Briefly, 0.5g root samples submitted to different ClO2 concentrations, immersed in a 10ml beaker with 0.4% TTC and 66mM sodium phosphate buffer. Samples were then put into graduated test tubes filled with10ml of methanol. Afterwards, the test tubes were left at 37°C for 4-7h in an incubator until the apical section turned completely white. Using a spectrophotometer for 485nm colorimetry. Root activity = amount of TTC reduction (μg)/fresh root weight (g) × time (h). Total N content in plant leaf, stem, root and soil was determined using an automatic kjeldahl apparatus (UDK159, VELP scientifica, Italy). N-efficiency parameters including NUE, UPE and UTE was calculated according to Zareabyaneh’s report [19].

2.3. RNA-seq analysis

Nine plants at bud stage in each group were randomly selected, three of which were pooled as a repeat and a total of three replicates were set for each group. The second leaf (the leaf under flag leaf) and root tissues were sampled for RNA-seq analysis using Illumina HiSeq 4000 platform. Three leaf (a) and root (b) samples were obtained from “X”, “Y” and “C”. Total RNA in each sample was purified using the n class="Chemical">cetyltrimethylammonium bromide (n class="Chemical">CTAB) method according to Thunyamada’s report [20]. Genomic DNA contamination were removed by DNase I (Takara, Tokyo, Japan). RNA concentration was determined using a NanoDrop ND-2100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Then, RNA sequencing library was constructed using a mRNA-seq Library Prep Kit for Illumina (Vazyme, Nanjing, Jiangsu, China). All the RNA sequencing libraries were then detected using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). In addition, the integrity of RNA was also determined by an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All these libraries were then subjected to the Illumina HiSeq 4000 platform followed by 2 × 150 bp paired-end sequencing.

2.4. Data processing and gene expression profile

Illumina CASAVA software (version 1.8.2, Illumina, Hayward, CA, USA) was used for converting the original image data to sequences by base calling. FastQC (version 0.11.5, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used for the quality control of raw data in the format of fastq. The adaptor and low-quality reads were then removed. HISAT software (version 2.0.4) [21] was used for mapping the clean data to the reference genome. Transcript assembly was conducted using cufflinks (version 2.1.1) [22], and the novel genes were identified using Cuffcompare in cufflinks (version 2.1.1) [22]. The fragments per kilobase of exon model per million mapped reads (FPKM) values of each read was calculated and the differentially expressed genes (DEn class="Gene">Gs) between two groups were identified usinpan>g DESeq (version 1.12.0) [23] with the criteria of padj < 0.05.

2.5. Enrichment analysis

The functional categories associated with DEn class="Gene">Gs were identified based on the en class="Gene">nrichment analysis. Gene Ontology (GO) biological processes (BPs) associated with DEGs were identified using GOseq [24]. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis was performed using the KOBAS (version 2.0) [25]. Terms with padj < 0.05 were considered as significant enrichment.

2.6. qRT-PCR analysis

The relative expression of several DEn class="Gene">Gs related to N metabolism were validated by qRT-PCR analysis. At bud stage, leaf (second leaf) and root RNA was isolated usinpan>g the n class="Chemical">CTAB method [20], and cDNA were synthesized as aforementioned. The relative expression were detected using the SHRR GREEN Mastermix (TaKaRa, Japan) and StepOnePlusTM Real-Time PCR System (Applied Biosystems, USA). The specific primers of the related genes were listed in S1 Table. The fold change of mRNA was calculated using the 2-ΔΔCt method by normalizing to the internal control gene EF1α.

2.7. Statistical analysis

Data were expressed as the mean ± SD (n = 3) and were subjected to the statistical analysis using GraphPad Prism 6 software. Statistical differences between two groups were analyzed using unpaired t-test, and that among six groups that were analyzed using the two-way ANOVA test followed by Tukey post-hoc. Correlation were analysis by Pearson correlation analysis. A probability value of less than 0.05 was considered a significant difference.

3. Results

3.1. N deficiency decreases potato growth

As expected, n class="Disease">N-deficient fertilization significantly decreased the fresh leaf weight (by about 40%), dry leaf weight (by about 20%), stem weight (by about 34%) and fresh tuber weight (by about 16%) inpan> all these three n class="Species">potato varieties (p < 0.01, Fig 1). We also found that the N ratio in leaf was significantly increased by N deficiency treatment (about 18%, p<0.01, Fig 2), but decreased in root and stem. The N contents (g/plant) in leaf, stem and root were lower in N-deficient group than that in N group. In addition, the UPE, UTE and NUE indexes were also significantly decreased by about 17%, 8% and 7% respectively by N deficiency in all these three potato varieties (Fig 2).
Fig 1

Statistical analysis of the growth performance parameters in response to by nitrogen (N) deficiency.

A, B and C shows the total fresh weight, total dry weight and tuber weight, respectively. N = 3, two-way ANOVA test followed by Tukey post-hoc, ** P <0.01 vs. corresponding variety under N treatment. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

Fig 2

Statistical analysis of the biochemistry indices of potato plant and soil in response to N deficiency.

A and B shows the N ratio (%) and N content (g/plant), respectively. C and D shows the total N (g/plant) and soil N (g/kg), respectively. E shows the value of NUE, UTE and UPE. NUE, N use efficiency; UPE, N uptake efficiency; UTE, N utilization efficiency; N = 3, two-way ANOVA test followed by Tukey post-hoc, ** P <0.01 vs. corresponding variety under N treatment. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

Statistical analysis of the growth performance parameters in response to by nitrogen (N) deficiency.

A, B and C shows the total fresh weight, total dry weight and tuber weight, respectively. N = 3, two-way ANOVA test followed by Tukey post-hoc, ** P <0.01 vs. corresponding variety under N treatment. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and n class="Disease">N-deficiency fertilizer, respectively.

Statistical analysis of the biochemistry indices of potato plant and soil in response to N deficiency.

A and B shows the N ratio (%) and N content (g/plant), respectively. C and D shows the total N (g/plant) and soil N (g/kg), respectively. E shows the value of n class="Chemical">NUE, UTE and UPE. n class="Chemical">NUE, N use efficiency; UPE, N uptake efficiency; UTE, N utilization efficiency; N = 3, two-way ANOVA test followed by Tukey post-hoc, ** P <0.01 vs. corresponding variety under N treatment. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

3.2. Influences of N deficiency on quality properties and enzyme activities

The results showed that the n class="Disease">N deficiency treatment significantly reduced the contents of soluble n class="Chemical">sugars and proteins in the leaves, but the effect was different in different developmental stages (Fig 3A and 3B). After N-deficiency treatment, cultivar C had the lowest soluble sugar (2.31±0.08, 0.76±0.03 and 0.76±0.03) and protein contents (1.18±0.03, 1.22±0.03 and 0.98±0.03) at the bud, tuber bulking and starch accumulation stages. Also, the root and GS activity of plants in Y (49.91±1.18 and 0.12±0.01), X (71.68±5.21 and 0.29±0.01) and C (63.78±5.21 and 0.23±0.01) at the seedling, tuber bulking and starch accumulation stages were significantly lower than that in Yn (72.37±5.21 and 0.18±0.01), Xn (96.00±2.26 and 0.33±0.01) and Cn (81.00±1.91 and 0.25±0.01), respectively (Fig 3C and 3E). Interestingly, N-deficient fertilization treatment significantly increased the NR activity (by about 25%) at the seedling stage, but decreased it (by about 16%) at the tuber bulking, starch accumulation and mature stages (Fig 3D). At the mature stage, the soluble sugars and proteins contents in leaf were low, which were not dependent on the application of N fertilizer, but on cultivars. N deficiency also decreased the NR (by ~16.55%) and GS activities (by ~15.06%) in potato leaves at the mature stage (Fig 3D and 3E).
Fig 3

Influence of N-deficiency fertilization on potato quality and enzyme activity.

(A), Soluble sugar content; (B), soluble protein content; (C), root activity; (D), NR activity; (E), GS activity. NR, nitrate reductase; GS, glutamine synthetase. Ss, Bs, Ts, Sa and Ms indicates the seedling, bud, tuber bulking, starch accumulation and mature stage, respectively. For statistical analysis, n = 3, two-way ANOVA test followed by Tukey post-hoc. Different letters indicate that mean values are significantly different (p<0.05). Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

Influence of N-deficiency fertilization on potato quality and enzyme activity.

(A), Soluble n class="Chemical">sugar content; (B), soluble proteinpan> content; (C), root activity; (D), n class="Gene">NR activity; (E), GS activity. NR, nitrate reductase; GS, glutamine synthetase. Ss, Bs, Ts, Sa and Ms indicates the seedling, bud, tuber bulking, starch accumulation and mature stage, respectively. For statistical analysis, n = 3, two-way ANOVA test followed by Tukey post-hoc. Different letters indicate that mean values are significantly different (p<0.05). Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

3.3. Summary of illumina sequencing

Illumina sequencing generated 1535.22 M clean reads and 241.49 G bases, with an average GC content and Q30 value of 41.45% and 93.84%, respectively (S2 Table). We identified 18, 90 and 1446 DEn class="Gene">Gs inpan> the leaf samples of cultivar C (Ca vs. CNa), X (Xa vs. XNa) and Y (Ya vs. YNa), respectively. For root samples, there were 0, 0 and 1009 DEn class="Gene">Gs found in cultivar C (Cb vs. CNb), X (Xb vs. XNb) and Y (Yb vs. YNb), respectively (Fig 4A and 4B). In leaves, the number of DEGs of pairwise comparison between varieties were showed in Fig 4C and 4E. The number of DEGs in root of pairwise comparison between varieties were showed in Fig 4D and 4F. Overall, N-deficiency treatment reduced the number of DEGs in the comparisons among different cultivars (Fig 4C and 4D).
Fig 4

Differentially expressed genes (DEGs) between different treatments.

A, the number of DEGs in the leaf (a for short) and root (b for short) induced by N deficiency. B, the Venn diagram of the DEGs in the leaf of potato in response to N deficiency. C and D, the statistics and Venn diagram of the DEGs in leaves of potato, respectively. E and F, the statistics and Venn diagram of the DEGs in root of potato, respectively. The red bar indicates up-regulated genes; green bar indicates down-regulated genes. A and b are short for leaf and root, respectively. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

Differentially expressed genes (DEGs) between different treatments.

A, the number of DEn class="Gene">Gs inpan> the leaf (a for short) anpan>d root (b for short) inpan>duced by pan> class="Disease">N deficiency. B, the Venn diagram of the DEGs in the leaf of potato in response to N deficiency. C and D, the statistics and Venn diagram of the DEGs in leaves of potato, respectively. E and F, the statistics and Venn diagram of the DEGs in root of potato, respectively. The red bar indicates up-regulated genes; green bar indicates down-regulated genes. A and b are short for leaf and root, respectively. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

3.4. N deficiency-induced DEGs associated with the N metabolism

The annotation results showed that most of the DEn class="Gene">Gs between CN and C were related to the processes of transport, n class="Chemical">nitrate transport and nitrogen compound transport (Fig 5A). StNRT 2.5 (PGSC0003DMG400016996), which was down-regulated in the leaf by N deficiency, act as a key regulator in variety C (Fig 5B). GO enrichment analysis showed that DEGs between YN and Y were significantly enriched in biological processes associated with N metabolism, including organonitrogen compound catabolic process, glutamine family amino acid metabolic process, arginine catabolic process to glutamate, and glutamate metabolic process (Fig 5C and S3 Table). DEGs like PGSC0003DMG400008356 (StGDH), PGSC0003DMG400023620 (StGS) and PGSC0003DMG400030984 (StCA) play key roles in these processes mentioned above. N deficiency treatment significantly decreased the expression of StGDH and StGS in the root of Y (Fig 5D and 5E). While StGS was increased by N deficiency treatment in leaves (Fig 5F). No N metabolism related GO terms were significantly enriched in X.
Fig 5

The biological processes associated with N metabolism in the leaf and root in three cultivars in response to N deficiency.

A, the biological processes associated with N metabolism in the leaf of cultivar C in response to N deficiency. B, the biological processes associated with N metabolism in the leaf and root of cultivar Y in response to N deficiency. The number ahead of the processes indicated gene number enriched in this process. N = 3, ** and * means p<0.01 and p<0.05, respectively. A and b are short for leaf and root, respectively. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

The biological processes associated with N metabolism in the leaf and root in three cultivars in response to N deficiency.

A, the biological processes associated with N metabolism in the leaf of cultivar C in response to n class="Disease">N deficiency. B, the biological processes associated with N metabolism inpan> the leaf and root of cultivar Y inpan> response to n class="Disease">N deficiency. The number ahead of the processes indicated gene number enriched in this process. N = 3, ** and * means p<0.01 and p<0.05, respectively. A and b are short for leaf and root, respectively. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

3.5. DEGs related to N metabolism among cultivars and between leaf and root

By comparing the transcriptome in different cultivars, many DEn class="Gene">Gs were identified among different cultivars after N containpan>inpan>g fertilizer treatment (Fig 4C and 4E). Unpan>der N containpan>inpan>g fertilization conditions, the up-regulated DEn class="Gene">Gs between XNa vs CNa were significantly enriched in “Photosynthesis”, “Porphyrin and chlorophyll metabolism”, “Carbon metabolism” and “Biosynthesis of secondary metabolites” (Table 1). While the down-regulated DEGs between XNa vs CNa were significantly enriched in “Mismatch repair”, “DNA replication”, “Plant hormone signal transduction”, “Nicotinate and nicotinamide metabolism” and “Valine, leucine and isoleucine degradation” (Table 1).
Table 1

The biological processes associated with the differentially expressed genes (DEGs) in the leaf of cultivar X versus cultivar C under N fertilization.

TermIDInput numberP-Value
Up-regulated DEGs
Photosynthesissot00195544.99E-10
Porphyrin and chlorophyll metabolismsot00860281.97E-06
Carbon fixation in photosynthetic organismssot00710387.38E-06
Glyoxylate and dicarboxylate metabolismsot00630347.50E-06
Carbon metabolismsot01200780.000287
Biosynthesis of amino acidssot01230720.000397
Glycine, serine and threonine metabolismsot00260300.000675
Photosynthesis-antenna proteinssot00196170.001225
Sulfur metabolismsot00920170.003874
Fructose and mannose metabolismsot00051230.005709
Glycolysis/Gluconeogenesissot00010390.006352
Metabolic pathwayssot011004390.00729
Biosynthesis of secondary metabolitessot011102420.008174
One carbon pool by folatesot00670100.008268
Carotenoid biosynthesissot00906150.012769
Zeatin biosynthesissot00908150.015266
Cyanoamino acid metabolismsot00460160.044154
Down-regulated DEGs
Mismatch repairsot03430214.03E-07
Homologous recombinationsot03440218.13E-06
DNA replicationsot03030201.25E-05
Nucleotide excision repairsot03420215.87E-05
beta-Alanine metabolismsot00410190.000597
Plant-pathogen interactionsot04626390.001005
Tryptophan metabolismsot00380110.003253
Plant hormone signal transductionsot04075480.005196
alpha-Linolenic acid metabolismsot00592130.006505
Nicotinate and nicotinamide metabolismsot0076070.007204
Protein processing in endoplasmic reticulumsot04141410.009618
Carotenoid biosynthesissot00906100.011613
Isoquinoline alkaloid biosynthesissot0095080.017799
Valine, leucine and isoleucine degradationsot00280140.017952
Ribosome biogenesis in eukaryotessot03008170.024065
Propanoate metabolismsot0064090.027304
Monoterpenoid biosynthesissot0090240.047538
Moreover, several N metabolism related DEn class="Gene">Gs were identified. For inpan>stance, the expression of ferredoxinpan>-n class="Gene">nitrite reductase (StNiR) gene in X were significantly lower than that in C in both the leaf and root tissues (Fig 6). In addition, many major facilitator superfamily (MFS) members were clustered, which showed different expression patterns between root and leaf in Y (Fig 6). Among these MFS members, there were several high affinity StNRTs, including StNRT2.4, StNRT2.5 and StNRT2.7 (Fig 6). These multiple expression profiles of MFS members suggested the nonredundant functions in potato leaf and root. However, the number of DEGs between cultivars under N deficiency conditions were fewer than that of under normal fertilization conditions in both leaf and root tissues (Figs 4 and 6). These DEGs were mainly involved in the processes of response to stresses and defense. These data showed that N deficiency induced stresses in all cultivars.
Fig 6

The expression profiles of the genes related to N metabolism in the leaf and root in response to N deficiency.

A and B shows the expression profile of N metabolism-associated genes in the leaf and root tissue, respectively. The redder the color, the higher the amount of expression; each of the different color blocks on the left represents a class of transcript clusters with similar expression levels. A and b are short for leaf and root, respectively. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

The expression profiles of the genes related to N metabolism in the leaf and root in response to N deficiency.

A and B shows the expression profile of N metabolism-associated genes in the leaf and root tissue, respectively. The redder the color, the higher the amount of expression; each of the different color blocks on the left represents a class of transcript clusters with similar expression levels. A and b are short for leaf and root, respectively. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and n class="Disease">N-deficiency fertilizer, respectively.

3.6. Validation of the N-metabolism related DEGs in leaf and root in response to N deficiency

The expression of Stn class="Gene">NRTs (2.4, 2.5 and 2.7), Stn class="Gene">NR, StNiR, StGDH, StGS and glutamine oxoglutarate aminotransferase (StGOGAT) in the leaf and root of the three cultivars were validated using qRT-PCR analysis (Fig 7). We confirmed that StNRT2.4 was increased in the leaf and root of all cultivars in response to N deficiency. However, StNRT2.5 andSt NRT2.7 in leaf were down-regulated by N deficiency. In the root, StNRT2.5 was up-regulated and StNRT2.7 was down-regulated in response to N deficiency, respectively. Two transcripts of StNiR (PGSC0003DMG400008262 and PGSC0003DMG400025823) had similar up-regulated profiles in the leaf, and were basically down-regulated in the root in response to N deficiency. Interestingly, two GDH transcripts had inverse expression profiles in response to N deficiency, one of which (PGSC0003DMG400008356) was up-regulated in the leaf and root of the three cultivars by N deficiency, but the other one (PGSC0003DMG400016001) showed a reverse pattern (Fig 7). Differential expression profiles were identified in transcripts coding GS and GOGAT. These results showed that the leaf and root of potato have different responses to N deficiency (Fig 7).
Fig 7

The expression profiles of the genes related to N metabolism in the leaf and root in response to N deficiency.

A and B shows the expression profile of N metabolism-associated genes in the leaf and root tissue, respectively. The redder the color, the higher the amount of expression; each of the different color blocks on the left represents a class of transcript clusters with similar expression levels. A and b are short for leaf and root, respectively. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and N-deficiency fertilizer, respectively.

A and B shows the expression profile of N metabolism-associated genes in the leaf and root tissue, respectively. The redder the color, the higher the amount of expression; each of the different color blocks on the left represents a class of transcript clusters with similar expression levels. A and b are short for leaf and root, respectively. Y, X and C indicate the cultivar of “Yanshu 4”, “Xiabodi” and “Chunshu 4”, respectively. N means treatment with N containing fertilizer (control). For example, YN and Y means “Yanshu 4” treated with N-containing and n class="Disease">N-deficiency fertilizer, respectively.

4. Discussion

n class="Species">Potato is the largest non-cereal food crop worldwide anpan>d ranpan>ked as the world’s fourth most importanpan>t food crop after pan> class="Species">rice, wheat, and maize. In a 100-gram portion, potato could provide 322 kilojoules of food energy and is a rich source of vitamin B6 and vitamin C (https://ndb.nal.usda.gov/ndb/search/list). N is an essential element for potato cultivation. Different varieties have different utilization efficiency of N fertilizer. The utilization efficiency of N of “Yanshu 4”, “Xiabodi” (cv. Shepody) and “Chunshu 4” potatoes are quite different in the production practice. Therefore, these three potato varieties are used as experimental objects. n class="Chemical">Nitrogen is the nutrient that’s most essential to plant growth. Plants use n class="Chemical">nitrogen to create their structure and to perform a range of functions. This chemical element is found in plant tissue, fruit, seeds and grains, and it forms part of chlorophyll, which is what makes plants green and allows them to process light into sugars [26]. N deficiency inhibited the growth and reduced the production in all the three potato cultivars. Our present study confirmed that N deficiency decreased the leaf weight, stem weight and the tuber production significantly. “Yanshu 4” had the highest production under both N-complete and N-deficient condition, followed by “Xiabodi” and “Chunshu 4”. We found that plants under N deficiency had higher UPE and NUE, but lower UTE compared with N-complete fertilization, which was consistent with Dai and Zhang’s reports [27,28]. Studies have shown that the root traits were important for N uptake. Commonly, the high N efficiency cultivars had higher root absorption, length, surface area, and volume than the low N efficiency cultivars, indicating that the above-mentioned root traits have improved dry matter production capacity under low n class="Chemical">nitrogen stress [27]. Inpan> the present study, N efficiencies were different among different cultivars: “Yanshu 4” had the highest UTE unpan>der n class="Disease">N deficiency, followed by “Chunshu 4”, indicating that genetic factors play an extremely important role in NUE. Transcriptome analysis showed that the leaf and root of cultivar “Yanshu 4” had the largest number of DEGs in response to N-deficiency stress compared with the other two cultivars, indicating that “Yanshu 4” is sensitive to nitrogen. An adequate supply of nitrogenous fertilizer may have excellent effects on “Yanshu 4”. Additionally, Jiao et al also identified “Yanshu 4” as a N-sensitive plant [29], which further confirmed our results. In these three varieties of our results, the different biochemical and gene expression profiles in response to nitrogen class="Disease">n deficiency were consistent with previous reports [30-32]. Jozefowicz et al [30] founpan>d that there are proteomic differences between two n class="Species">potato cultivars in response to N deficiency. Also, they reported the different activation of the GS/GOGAT pathway between the two potato cultivars. Interestingly, Jozefowicz et al found that the GDH was up-regulated in both the N-deficiency tolerant and sensitive potato cultivars under N-deficiency conditions, while GS was up-regulated only in the N tolerant potato cultivar by N deficiency [30]. Tiwari’s report concluded that many potential genes play very crucial roles in N stress tolerance [32]. The difference is that this study used three potato varieties with different resistance to N deficiency for transcriptome studies. According to the present study, Yanshu4 increased StGAD, StGDH and StGS, and reduced StNRT2.5 and StNRT2.7 in response to N deficiency. The regulation of these genes in the other two varieties was not obvious. Jozefowicz’s research showed similar results with our findings [30], but the difference is that the N deficiency has a more extensive influence on the gene expression profile than protein level of high-resistant varieties. In comparison with sufficient N fertilization, N deficiency induced a large number of DEGs in the leaf and root of cultivar “Yanshu 4”. However, only a few DEGs were identified in the cultivar “Xiabodi” and “Chunshu 4”. Different varieties of potato showed different gene expression profiles to N stress. Two possible causes were considered, one is that there might be a lot of frontloaded genes in the two cultivars as has been found in other species [33,34]; another possibility is that they were less affected by N deficiency. Although the former seems more credible, further research is needed to verify. These results suggested that the mechanisms in potato in response to N deficiency were cultivar-dependent. At the genetic level, the different expression patterns of genes in response to N deficiency were determined by gene diversity, which might also be the root cause of different varieties of potatoes having different responses to N deficiency. How to make good use of these excellent genetic resources for cross breeding was very worthy of our future research, and “Yanshu 4” might be an excellent candidate breeding resource. Of all these DEn class="Gene">Gs response to n class="Disease">N deficiency in “Yanshu 4”, some key genes (associated with N metabolism) were up-regulated in both leaf and root, such as StGAD, StGDH, StGS. Also, some down-regulated DEGs like StNRT2.5 and StNRT2.7 were also act as key regulators. This result was in line with the study of Gálvez et al [31], who identified 39 common N responsive genes in response to N deficiency, including NiR, GOGAT, GS and GAD. It has been reported that the increased GS is correlated with elevated grain yield and NUE [35,36]. StGS is an essential enzyme crucial for ammonium assimilation and N remobilization. It assimilates ammonium into the amide position of glutamine (Fig 8) [37]. In addition, the increased NR activity also correlated with enhanced NUE in cotton under N deficiency [38,39]. Our biochemical experiments also showed the positive relationship between the NR activity and NUE. The NR activity was decreased by N deficiency, and the decreased UTE was in line with the decreased NR activity. Moreover, the increased StGS gene in response to N deficiency was correlated with the increased UTE in “Yanshu 4”, but contrary to the decreased NUE and UPE. These data indicated that the NUE and growth of potato under N deficiency was not unilaterally determined by these two factors.
Fig 8

A schematic representation of the N metabolism pathway emphasizing the differentially expressed genes in potato.

NRT, nitrate transporter; NR, nitrate reductase; NiR, nitrite reductase; GS, glutamine synthetase; GOGAT, glutamate synthase; AFMID, formamidase; CA, carbonic anhydrase; CYN, cyanate hydratase; GDH, glutamate synthase.

A schematic representation of the N metabolism pathway emphasizing the differentially expressed genes in potato.

n class="Gene">NRT, pan> class="Chemical">nitrate transporter; NR, nitrate reductase; NiR, nitrite reductase; GS, glutamine synthetase; GOGAT, glutamate synthase; AFMID, formamidase; CA, carbonic anhydrase; CYN, cyanate hydratase; GDH, glutamate synthase. Our present study identified that several n class="Chemical">nitrate transporter codinpan>g genes were downpan>-regulated by n class="Disease">N deficiency including StNRT2.5, StNRT2.7, and StNiR in potato leaf, while StNRT2.5 in root were up-regulated after N-deficiency treatment. These DEGs were differentially expressed among cultivars, the difference in promoter or copy number might be the two key reasons for the differential response and regulation of in three cultivars of potato. Tiwari et al reported that most of the StNRT family members were down-regulated in roots under low N conditions [32], which was consisted with our results. The NRTs, NR and NiR are crucial for the acquiring N and its conversion to ammonia (Fig 8) [40]. NRT2 family is known to control N uptake and transport and is widely distributed in plants [41]. Lezhneva et al [40] reported that the AtNRT2.5 was expressed in the shoot and root of Arabidopsis in response to N deficiency. It plays a role in obtaining N elements [40]. Arabidopsis has seven NRT2 family members, and NRT2.7 is the only NRT2 member located on the tonoplast membrane in the seeds, and it interacts with NAR2.1 during nitrate transport [42,43]. However, the expression profiles of StNRT2.4, StNiR, and StNR were increased in potato leaf by N deficiency, which suggested the increased N metabolism.

5. Conclusions

Nitrogen class="Disease">n deficiency inpan>duced a large number of DEn class="Gene">Gs in the leaf and root of cultivar “Yanshu 4”, and only few DEGs in the cultivar “Xiabodi” and “Chunshu 4”. The mechanisms in potato in response to N deficiency were cultivar-dependent. “Yanshu 4” might be an excellent candidate cross breeding resource. The up-regulated DEGs related to N metabolism were correlated with increased UTE in potato in response to N deficiency, but the association of them with NUE needs further investigation. In addition, the decreased StNRT2.4, StNiR, and StNR by N deficiency indicated the increased N metabolism in potato. This study will provide data support for the breeding of high N utilization efficiency potatoes in Northeast China.

The sequences of primers used for the PCR analysis.

(DOCX) Click here for additional data file.

RNA-seq data summary and quality analysis.

(DOCX) Click here for additional data file.

Several key enzymes differentially expressed by N deficiency and between cultivars and associated with the N metabolism.

(DOCX) Click here for additional data file. 12 May 2020 n class="Chemical">PONE-D-20-07306 Nitrogen class="Disease">n deficiency inpan>duced alterations inpan> leaf anpan>d root of three cultivars of pan> class="Species">potato (Solanum tuberosum L.) PLOS ONE Dear Dr. Han, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We would appreciate receiving your revised manuscript by 30 May 2020. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Mayank Gururani Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Partly ********** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ********** 3. Have the authors made all data underlying the findinn class="Gene">gs inpan> their manpan>uscript fully available? The PLOS Data policy requires authors to make all data underlying the findinn class="Gene">gs described inpan> their manpan>uscript fully available without restriction, with rare exception (please refer to the Data Availability Statemenpan>t inpan> the manpan>uscript PDF file). The data should be provided as part of the manpan>uscript or its supportinpan>g inpan>formation, or deposited to a public repository. For example, inpan> addition to summary statistics, the data poinpan>ts behinpan>d meanpan>s, medianpan>s anpan>d varianpan>ce measures should be available. If there are restrictions on publicly sharinpan>g data—e.g. pan> class="Species">participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ********** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: No ********** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The study presents unique and original data that will significantly contribute to our understanding on the molecular underpinninn class="Gene">gs of n class="Chemical">nitrogen responses in plants particularly on potato. While it has already been reported by Jozefowicz et al. (2017), nitrogen deficiency does induce alterations in root proteome of potato varieties contrasting their response to low N. Just recently, Tiwari et al. (2020) presented their results on the transcriptome analysis of potato shoots, roots, and stolons under nitrogen stress. This paper by Zhang et al. investigates the association of transcriptomic profiles with the efficiencies of N using 3 potato cultivars. This study will have a bigger impact if the authors discuss the implications of their results in relation to genetic variability. Unfortunately, the paper is poorly written. Presentation of ideas and concepts are hard to follow as it lacks the logical flow for the entire manuscript to be comprehensible. Although the experiments and statistical analysis have indeed been performed to high technical standard, however, further analysis of their data are still needed and presented for discussion and elaborated in their conclusion. Unfortunately, the conclusion is barely comprehensible as it only reiterates their findinn class="Gene">gs without discussinpan>g any implications. Also, may I suggest the title to include the term “Transcriptome” as the entire paper revolves around transcriptomic analysis of n class="Species">potato cultivars unpan>der nitrogepan> class="Disease">n deficiency. Reviewer #2: The manuscript by Zhang et al. investigates the role of n class="Chemical">nitrogen (N) inpan> pan> class="Species">potato (Solanum tuberosum) plants, and in particular on three different cultivars (CVs): Yanshu 4, Xiabodi and Chunshu 4. The plants were treated with N-sufficient or with N-deficient fertilization. In the work, morphological (plant growth and tuber yield), biochemical (content soluble sugars, proteins, nitrate reductase and glutamine synthetase activities) and biomolecular parameters were evaluated. The research is quite interesting, however some revisions are necessary before publication. First of all, I suggest to the authors a substantial revision of the English, since there are many spelling mistakes in the text. I could not make corrections of all of them. I suggest to the author to send the manuscript to a native English speaker before submission. At the LINE 21 – 27 of the ABSTRACT, there is a simple list of methods used in the work, without any results and discussion. Moreover, is not clear what authors mean with the sentence “and root were detected” (LINE 23). I suggest to re-write the abstract, highlighting better the results. In the present version, it is too descriptive. The INTRODUCTION is well written, and rigidly follows the discussion of some important issues. It initially introduces the importance of n class="Species">potato from a nutritional and agronomic poinpan>t of view, and then discusses inpan>to details the mainpan> problems linpan>ked to the cultivation of the tuber, inpan>cludinpan>g N levels. Inpan> this regard, the authors also describe the different practices inpan> use for the improvement of n class="Chemical">NUE. However, I would suggest to the authors to add some additional information about the cultivars used in this work. Is there a specific reason related to the use of these selected CVs? Were they chosen for economical, nutritional, genetic or what? This information should be added to the last part of the introduction. MATERIALS and METHODS: sometimes the methods are not well described or reported. I suppose the authors used qRT-PCR to validate the RNA-seq analysis. A couple of times they used PCR (also in the abstract) not qRT-PCR. I would suggest to add EC numbers for n class="Gene">nitrate reductase and n class="Gene">glutamine synthetase and also to provide some additional information about enzymatic activity measurements, although a kit was used. I don’t understand the unit used for enzyme activity calculation (see Figure 1). Moreover, what does it mean “root activity”? In the RESULTS section, several information are missed, and must be implemented. I think it is not strictly necessary reporting numerical values in the manuscript text if present in the tables, however, at least the variation of the trend must be described (e.g. % of the change among the different CVs or between the two treatments). On the other hand, when data are expressed as Figures, it is necessary to report in the text the most important and representative numerical values (means ± standard deviation), and describe the changes among CVs and treatments. The DISCUSSION is well motivated, but is limited only to the data obtained in this work. This part must be expanded. I suggest to introduce a short initial part, in which the importance of nutritional and economical aspects of n class="Species">potato are discussed inpan> relation with the three different CVs employed. Inpan> addition, the importance of N, and its n class="Disease">deficiency in plant nutrition has to be introduced. The CONCLUSIONS are too synthetic and don’t highlight the obtained results. I suggest to expand also this section by explaining the importance of the research and the future applications. LEGENDS FOR FIGURES: they must be improved. The authors should report as much information as possible. For example the statistical treatment used and the meaning of the letters on the bars (Figure 1). The legend must be clear for the reader ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. 20 Jul 2020 Dear Editor, Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Nitrogen class="Disease">n deficiency inpan>duced alterations inpan> leaf and root of three cultivars of n class="Species">potato (Solanum tuberosum L.)”. Those comments are all valuable. We have studied comments carefully and made corrections. The revised manuscript is highlighted in Tracked Changes version. Point-by-point response to the reviewer’s comments Reviewer reports: Reviewer 1: The study presents unique and original data that will significantly contribute to our understanding on the molecular underpinninn class="Gene">gs of n class="Chemical">nitrogen responses in plants particularly on potato. While it has already been reported by Jozefowicz et al. (2017), nitrogen deficiency does induce alterations in root proteome of potato varieties contrasting their response to low N. Just recently, Tiwari et al. (2020) presented their results on the transcriptome analysis of potato shoots, roots, and stolons under nitrogen stress. This paper by Zhang et al. investigates the association of transcriptomic profiles with the efficiencies of N using 3 potato cultivars. This study will have a bigger impact if the authors discuss the implications of their results in relation to genetic variability. Unfortunately, the paper is poorly written. Presentation of ideas and concepts are hard to follow as it lacks the logical flow for the entire manuscript to be comprehensible. Although the experiments and statistical analysis have indeed been performed to high technical standard, however, further analysis of their data are still needed and presented for discussion and elaborated in their conclusion. Unfortunately, the conclusion is barely comprehensible as it only reiterates their findinn class="Gene">gs without discussinpan>g any implications. Response: Thank you for your comments. Firstly, we accept the suggestion to try our best to improve the English language. To further improve the quality of written English, the manuscript has also been edited by Caughman Corey, who was a native English speaker. Then, we have reorganized the manuscript and hope that the new manuscript will be more logical. In addition, to better present the results, we have made major revisions to the results section. New data were added. Also, we re-drawn figure 3 and added N-related gene expression results (Figure 4). Importantly, we have revised the discussion and conclusion sections, hoping that the newly submitted manuscript will be improved. Also, may I suggest the title to include the term “Transcriptome” as the entire paper revolves around transcriptomic analysis of n class="Species">potato cultivars unpan>der nitrogepan> class="Disease">n deficiency. Response: Thank you for your comments. We re-titled the manuscript to “Transcriptome analysis reveals Nitrogen class="Disease">n deficiency inpan>duced alterations inpan> leaf anpan>d root of three cultivars of pan> class="Species">potato (Solanum tuberosum L.)” Reviewer 2: First of all, I suggest to the authors a substantial revision of the English, since there are many spelling mistakes in the text. I could not make corrections of all of them. I suggest to the author to send the manuscript to a native English speaker before submission. Response: Thank you for your comments. We accept the suggestion to try our best to improve the English language. To further improve the quality of written English, the manuscript has also been edited by Caughman Corey, who was a native English speaker. At the LINE 21 – 27 of the ABSTRACTS, there is a simple list of methods used in the work, without any results and discussion. Moreover, is not clear what authors mean with the sentence “and root were detected” (LINE 23). I suggest to re-write the abstract, highlighting better the results. In the present version, it is too descriptive. Response: Thank you for your comments. We re-write the abstract section and highlight the main results. The reorganized abstract is improved. The INTRODUCTION is well written, and rigidly follows the discussion of some important issues. It initially introduces the importance of n class="Species">potato from a nutritional and agronomic poinpan>t of view, and then discusses inpan>to details the mainpan> problems linpan>ked to the cultivation of the tuber, inpan>cludinpan>g N levels. Inpan> this regard, the authors also describe the different practices inpan> use for the improvement of n class="Chemical">NUE. However, I would suggest to the authors to add some additional information about the cultivars used in this work. Is there a specific reason related to the use of these selected CVs? Were they chosen for economical, nutritional, genetic or what? This information should be added to the last part of the introduction. Response: Thank you for your comments. The utilization efficiency of N of “Yanshu 4”, “Xiabodi” (cv. Shepody) and “Chunshu 4” n class="Species">potatoes are quite differenpan>t inpan> the productionpan> practice. Therefore, these three pan> class="Species">potato varieties are used as experimental objects. MATERIALS and METHODS: sometimes the methods are not well described or reported. I suppose the authors used qRT-PCR to validate the RNA-seq analysis. A couple of times they used PCR (also in the abstract) not qRT-PCR. I would suggest to add EC numbers for n class="Gene">nitrate reductase and n class="Gene">glutamine synthetase and also to provide some additional information about enzymatic activity measurements, although a kit was used. I don’t understand the unit used for enzyme activity calculation (see Figure 1). Moreover, what does it mean “root activity”? Response: Thank you for your comments. All the PCR mentioned in MATERIALS and METHODS should be qRT-PCR. We have revised the related parts. The EC number for n class="Gene">nitrate reductase anpan>d pan> class="Gene">glutamine synthetase was provide as required. The function of root activity was added in the method section. For the unit, we revised it in the newly submitted manuscript. In the RESULTS section, several information are missed, and must be implemented. I think it is not strictly necessary reporting numerical values in the manuscript text if present in the tables, however, at least the variation of the trend must be described (e.g. % of the change among the different CVs or between the two treatments). On the other hand, when data are expressed as Figures, it is necessary to report in the text the most important and representative numerical values (means ± standard deviation), and describe the changes among CVs and treatments. Response: Thank you for your comments. We added some main information to the manuscript. But it is un class="Gene">nrealistic to fully describe the ranpan>ge of inpan>crease or decrease, especially the contenpan>t inpan> the table. We have two treatmenpan>ts for 3 varieties, so the ranpan>ge of variation is diverse. Evenpan> the chanpan>ginpan>g trenpan>ds of differenpan>t varieties are also differenpan>t betweenpan> differenpan>t treatmenpan>ts. Therefore, we only added the mainpan> contenpan>t. The DISCUSSION is well motivated, but is limited only to the data obtained in this work. This part must be expanded. I suggest to introduce a short initial part, in which the importance of nutritional and economical aspects of n class="Species">potato are discussed inpan> relation with the three different CVs employed. Inpan> addition, the importance of N, and its n class="Disease">deficiency in plant nutrition has to be introduced. Response: Thank you for your comments. We added the importance of nutritional and economical aspects of n class="Species">potato. The utilization efficiency of N of “Yanshu 4”, “Xiabodi” (cv. Shepody) and “Chunpan>shu 4” n class="Species">potatoes are quite different in the production practice. Therefore, these three potato varieties are used as experimental objects. The importance of N was also discussed in the newly submitted manuscript. The CONCLUSIONS are too synthetic and don’t highlight the obtained results. I suggest to expand also this section by explaining the importance of the research and the future applications. Response: Thank you for your comments. We rewrote our conclusions, summarizing the important results of this study and the importance of this study for n class="Species">potato breedinpan>g. LEGENDS FOR FIGURES: they must be improved. The authors should report as much information as possible. For example the statistical treatment used and the meaning of the letters on the bars (Figure 1). The legend must be clear for the reade Response: Thank you for your comments. All the legends of figures and tables were improved. Submitted filename: Point-by-point response.docx Click here for additional data file. 17 Aug 2020 n class="Chemical">PONE-D-20-07306R1 Transcriptome analysis reveals Nitrogen class="Disease">n deficiency inpan>duced alterations inpan> leaf anpan>d root of three cultivars of pan> class="Species">potato (Solanum tuberosum L.) PLOS ONE Dear Dr. Han, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by 31 August 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Mayank Gururani Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: (No Response) Reviewer #2: All comments have been addressed ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes ********** 4. Have the authors made all data underlying the findinn class="Gene">gs inpan> their manpan>uscript fully available? The PLOS Data policy requires authors to make all data underlying the findinn class="Gene">gs described inpan> their manpan>uscript fully available without restriction, with rare exception (please refer to the Data Availability Statemenpan>t inpan> the manpan>uscript PDF file). The data should be provided as part of the manpan>uscript or its supportinpan>g inpan>formation, or deposited to a public repository. For example, inpan> addition to summary statistics, the data poinpan>ts behinpan>d meanpan>s, medianpan>s anpan>d varianpan>ce measures should be available. If there are restrictions on publicly sharinpan>g data—e.g. pan> class="Species">participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This paper by Zhang et al. investigates the association of transcriptomic profiles with n class="Disease">N deficiency usinpan>g 3 n class="Species">potato cultivars. This study will have a bigger impact if the authors discuss the implications of their results in relation to genetic variability which was barely discussed. Similarly, it is best to include in their discussion on how their results differ or add up to those already contributed by Jozefowicz et al. (2017) and Tiwari et al. (2020). Moreover, the conclusion needs to be further improved and expounded as it remains shallow reiterating their findings but without value/importance. Statistical analyses only mentioned unpaired t-test and two-way ANOVA in the Methods while there is a plethora of statistical analyses (Holm- Sidak, Tukey’s Test, etc) which are more appropriate for this study. How was the correlation analysis done to conclude positive/negative correlation? The paper needs to be presented in a more intelligible manner (scientific jargon not just standard English). The following lines/sentences needs to be rephrased or improved: Lines: 49, 69, 74-75, 94-97, 102-104, 179-188, 199-201, 208-214, 220-225,228-231, 237-239, 243-251,270-271, 275-276, 284-288, 309-311, 354, 370, 374-376 Others: Line 28 what is correlation relationship? Isn’t this positive or negative correlation? Line 79 Delete “in April 2017” Line 110 RNA-seq analysis using ___ (brand & model of equipment) Line 111 delete as YNa, YNb ….. Line 216 Delete the terms “statistics of the” Tables 1 and 2 need to be presented as graphs in figure with error bars representing SD. Line 251 N containing fertilizer (not contained) Line 299-300 what is Pn class="Gene">GSC… and Pn class="Gene">GSC…? Line 323 start new paragraph with, “n class="Chemical">Nitrogen is the nutrienpan>t… Line 330 what is N-colplete fertilization? Line 331 start new paragraph with, “Studies have shown …. Reviewer #2: The manuscript has been improved and most part of my comments were properly addressed. However, some revision is still necessary. Line 28: n class="Gene">GS anpan>d pan> class="Gene">NR must be written with the full names as well. The same for StGDH, StGS and StCA (Line 30) and MFS members, like StNRT2.4, StNRT2.5 and StNRT2.7 (Line 33) Line 50. Its tuber is used not tuber of which Line 116. I would write the n class="Chemical">anthrone method anpan>d pan> class="Chemical">Coomassie Brilliant Blue G-250 dye-binding method were used… Line 117. To detect not to detected Line 118. I think that it is better to report the plural. Soluble n class="Chemical">sugars anpan>d soluble proteinpan>s. Line 125: which kind of n class="Chemical">phosphate buffer? K or Na? I thinpan>k the followinpan>g senpan>tenpan>ce is unpan>clear: Thenpan> the color reaction is carried out, anpan>d finpan>ally the absorbanpan>ce is measured to determinpan>e the enpan>zyme activity. Better to rewrite the senpan>tenpan>ce anpan>d use the past tenpan>se. Line 132. RNAseq analysis since seq is already sequencing Line 164. n class="Chemical">CTAB: report the full name as well. Methods should be singular, so method Line 207. n class="Chemical">Sugars anpan>d proteinpan>s, not pan> class="Chemical">sugar and protein Line 243. Respectively, not respectiverly Line 261. Figure legend. Bar not bat Line 270. DEG not DEn class="Gene">Gs Line 318. N metabolism not N metabolisms Line 392. N-completed not N-colplete Line 416. DEG not DEn class="Gene">Gs Supplementary Table S1: qRT-PCR not PCR ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 30 Aug 2020 Dear editor: Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Transcriptome analysis reveals Nitrogen class="Disease">n deficiency inpan>duced alterations inpan> leaf and root of three cultivars of n class="Species">potato (Solanum tuberosum L.)”. Those comments are all valuable. We have studied comments carefully and made corrections. The revised manuscript is highlighted in Tracked Changes version. Point-by-point response to the reviewer’s comments Response: This paper by Zhang et al. investigates the association of transcriptomic profiles with n class="Disease">N deficiency usinpan>g 3 pan> class="Species">potato cultivars. This study will have a bigger impact if the authors discuss the implications of their results in relation to genetic variability which was barely discussed. Response: Thank you for your comments. The discussion section was improved. At the genetic level, the different expression patterns of genes in response to n class="Disease">N deficiency were determinpan>ed by gene diversity, which might also be the root cause of different varieties of n class="Species">potatoes having different responses to N deficiency. How to make good use of these excellent genetic resources for cross breeding was very worthy of our future research, and “Yanshu 4” might be an excellent candidate breeding resource. This part of the content has been added in the discussion. Similarly, it is best to include in their discussion on how their results differ or add up to those already contributed by Jozefowicz et al. (2017) and Tiwari et al. (2020). Response: Thank you for your comments. Jozefowicz’s research showed similar results with our findinn class="Gene">gs, but the difference is that the n class="Disease">N deficiency has a more extensive influence on the gene expression profile than protein level of high-resistant varieties. The experimental design of Tiwari’s research is also different from this study, but some of the results are consistent. In the discussion, we added a part of the content to explain the difference between this study and previous studies, but more importantly, discuss the possible reasons that caused the results of this study Moreover, the conclusion needs to be further improved and expounded as it remains shallow reiterating their findinn class="Gene">gs but without value/importanpan>ce. Response: Thank you for your comments. We have condensed the conclusion part to make the conclusion more prominent. Statistical analyses only mentioned unpaired t-test and two-way ANOVA in the Methods while there is a plethora of statistical analyses (Holm- Sidak, Tukey’s Test, etc) which are more appropriate for this study. Response: Thank you for your comments. The data were analyzed using the ANOVA test followed by Tukey post-hoc. We missed the test method in the previous version, this time we added it. How was the correlation analysis done to conclude positive/negative correlation? Response: Thank you for your comments. Correlation were analysis by Pearson correlation analysis. The paper needs to be presented in a more intelligible manner (scientific jargon not just standard English). The following lines/sentences needs to be rephrased or improved: Lines: 49, 69, 74-75, 94-97, 102-104, 179-188, 199-201, 208-214, 220-225,228-231, 237-239, 243-251,270-271, 275-276, 284-288, 309-311, 354, 370, 374-376 Response: Thank you for your comments. All the mentioned parts were revised as required. Others: Line 28 what is correlation relationship? Isn’t this positive or negative correlation? Line 79 Delete “in April 2017” Line 110 RNA-seq analysis using ___ (brand & model of equipment) Line 111 delete as YNa, YNb ….. Line 216 Delete the terms “statistics of the” Tables 1 and 2 need to be presented as graphs in figure with error bars representing SD. Line 251 N containing fertilizer (not contained) Line 299-300 what is Pn class="Gene">GSC… and Pn class="Gene">GSC…? Line 323 start new paragraph with, “n class="Chemical">Nitrogen is the nutrienpan>t… Line 330 what is N-colplete fertilization? Line 331 start new paragraph with, “Studies have shown …. Response: Thank you for your comments. The mentioned parts were all revised as required. Reviewer #2: The manuscript has been improved and most part of my comments were properly addressed. However, some revision is still necessary. Line 28: n class="Gene">GS anpan>d pan> class="Gene">NR must be written with the full names as well. The same for StGDH, StGS and StCA (Line 30) and MFS members, like StNRT2.4, StNRT2.5 and StNRT2.7 (Line 33) Line 50. Its tuber is used not tuber of which Line 116. I would write the n class="Chemical">anthrone method anpan>d pan> class="Chemical">Coomassie Brilliant Blue G-250 dye-binding method were used… Line 117. To detect not to detected Line 118. I think that it is better to report the plural. Soluble n class="Chemical">sugars anpan>d soluble proteinpan>s. Line 125: which kind of n class="Chemical">phosphate buffer? K or Na? I thinpan>k the followinpan>g senpan>tenpan>ce is unpan>clear: Thenpan> the color reaction is carried out, anpan>d finpan>ally the absorbanpan>ce is measured to determinpan>e the enpan>zyme activity. Better to rewrite the senpan>tenpan>ce anpan>d use the past tenpan>se. Line 132. RNAseq analysis since seq is already sequencing Line 164. n class="Chemical">CTAB: report the full name as well. Methods should be singular, so method Line 207. n class="Chemical">Sugars anpan>d proteinpan>s, not pan> class="Chemical">sugar and protein Line 243. Respectively, not respectiverly Line 261. Figure legend. Bar not bat Line 270. DEG not DEn class="Gene">Gs Line 318. N metabolism not N metabolisms Line 392. N-completed not N-colplete Line 416. DEG not DEn class="Gene">Gs Response: Thank you for your approval and comments. All the mentioned parts in the manuscript was revised as required. Supplementary Table S1: qRT-PCR not PCR Response: Thank you for your comments. We revised it as required. Submitted filename: Response.docx Click here for additional data file. 14 Sep 2020 n class="Chemical">PONE-D-20-07306R2 Transcriptome analysis reveals Nitrogen class="Disease">n deficiency inpan>duced alterations inpan> leaf anpan>d root of three cultivars of pan> class="Species">potato (Solanum tuberosum L.) PLOS ONE Dear Dr. Han, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by 27th Sept. 2020. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Mayank Gururani Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed Reviewer #3: (No Response) ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: Yes Reviewer #3: (No Response) ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: Yes Reviewer #3: (No Response) ********** 4. Have the authors made all data underlying the findinn class="Gene">gs inpan> their manpan>uscript fully available? The PLOS Data policy requires authors to make all data underlying the findinn class="Gene">gs described inpan> their manpan>uscript fully available without restriction, with rare exception (please refer to the Data Availability Statemenpan>t inpan> the manpan>uscript PDF file). The data should be provided as part of the manpan>uscript or its supportinpan>g inpan>formation, or deposited to a public repository. For example, inpan> addition to summary statistics, the data poinpan>ts behinpan>d meanpan>s, medianpan>s anpan>d varianpan>ce measures should be available. If there are restrictions on publicly sharinpan>g data—e.g. pan> class="Species">participant privacy or use of data from a third party—those must be specified. Reviewer #2: Yes Reviewer #3: (No Response) ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: Yes Reviewer #3: (No Response) ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: (No Response) Reviewer #3: Overall the paper is well-written, and provides valuable insight on transcriptome analysis reveals n class="Disease">N-deficiency inpan> three cultivars of n class="Species">potato (Solanum tuberosum L.). Below are suggestions and comments to improve the clarity and message of the manuscript. >1.The authors choose three cultivars n class="Species">potato, Yanpan>shu 4, Xiabodi anpan>d Chunpan>shu 4. How did the authors decide three cultivars? From cultivar resources? There was different genetic background in three cultivars n class="Species">potato, the differenpan>ce inpan> the growth performanpan>ce parameters may be not onpan>ly affected by N-related genpan>es. So, the hybrids offsprinpan>g of two differenpan>t varieties were more reasonpan>able. >2. Unclear statistics: you mention three plants for each replicate have been used, the number of n should be used for statistical evaluation, which tests were applied in legends? >3.In fig4 C D, why the DEn class="Gene">Gs were less unpan>der pan> class="Disease">N-deficiency condition in cultivars. There may be a lot frontloaded genes in cultivars. Reference: Barshis etal, 2013 “Genomic basis for coral resilience to climate change” >4. In RNA-seq analysis: why choose the second leaf? not flag leaf? And in RT-qPCR, which leaf was used, growth stage? >5. In Fig6 and 7, It would be two reasons about the differential response and regulation of in three cultivars of n class="Species">potato, promoter or copy number. The expression of one genpan>e was not usually compared among differenpan>t cultivars by RT-qPCR. ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: No Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 29 Sep 2020 Dear editor: Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “Transcriptome analysis reveals Nitrogen class="Disease">n deficiency inpan>duced alterations inpan> leaf and root of three cultivars of n class="Species">potato (Solanum tuberosum L.)”. Those comments are all valuable. We have studied comments carefully and made corrections. The revised manuscript is highlighted in Tracked Changes version. Point-by-point response to the reviewer’s comments Review Comments to the Author Reviewer #2: (No Response) Reviewer #3: Overall the paper is well-written, and provides valuable insight on transcriptome analysis reveals n class="Disease">N-deficiency inpan> three cultivars of n class="Species">potato (Solanum tuberosum L.). Below are suggestions and comments to improve the clarity and message of the manuscript. >1.The authors choose three cultivars n class="Species">potato, Yanpan>shu 4, Xiabodi anpan>d Chunpan>shu 4. How did the authors decide three cultivars? From cultivar resources? There was different genetic background in three cultivars n class="Species">potato, the differenpan>ce inpan> the growth performanpan>ce parameters may be not onpan>ly affected by N-related genpan>es. So, the hybrids offsprinpan>g of two differenpan>t varieties were more reasonpan>able. Response: Thank you for your comments. In the production practice, we found that the utilization efficiency of N of “Yanshu 4”, “Xiabodi” (cv. Shepody) and “Chunshu 4” n class="Species">potatoes are quite different inpan> the production practice. Accordinpan>g to production experience, unpan>der the condition of sufficient n class="Chemical">nitrogen fertilizer, “Yanshu 4” was a high-absorption but low-utilization potato, “Xiabodi” was a medium-absorption and medium-utilization potato, and “Chunshu 4” was a low-absorption but high-utilization potato (lines 72-75). The aim of this study was to reveal the transcriptional responses of different cultivars to N deficiency under different conditions. In addition, you encourage us to hybrid offspring of two different varieties, this is very important for our follow-up study. We are willing to accept your opinion to carry out hybridization experiment and reveal the location of functional genes and QTLs associated with the N-utilization efficiency. Thanks again for the above comment and suggestion. >2. Unclear statistics: you mention three plants for each replicate have been used, the number of n should be used for statistical evaluation, which tests were applied in legends? Response: Thank you for your comments. We added the number of replicates in the statistical analysis section. Also, the tests were also added in the legends of Figure 1, 2 and 3. >3. In fig4 C D, why the DEn class="Gene">Gs were less unpan>der pan> class="Disease">N-deficiency condition in cultivars. There may be a lot frontloaded genes in cultivars. Reference: Barshis etal, 2013 “Genomic basis for coral resilience to climate change” Response: Thank you for your comments. We accept your suggestion and discuss it in the revised manuscript (Discussion section, lines 361-362). >4. In RNA-seq analysis: why choose the second leaf? not flag leaf? And in RT-qPCR, which leaf was used, growth stage? Response: Thank you for your comments. Because of individual differences, the flag leaf size is different. To avoid the impact of this difference, we used the second leaf. For qRT-PCR analysis, the second leaf was also used. Samples were collected at bud stage (line 149). >5. In Fig6 and 7, It would be two reasons about the differential response and regulation of in three cultivars of n class="Species">potato, promoter or copy number. The expression of one genpan>e was not usually compared among differenpan>t cultivars by RT-qPCR. Response: Thank you for your comments. We added the reason in discussion section. Also, we have modified the figures to reflect intra species comparisons rather than inter variety comparisons (lines 379-379). Thank you again for your comments, which is very helpful to improve our article. Submitted filename: Response.docx Click here for additional data file. 1 Oct 2020 Transcriptome analysis reveals Nitrogen class="Disease">n deficiency inpan>duced alterations inpan> leaf anpan>d root of three cultivars of pan> class="Species">potato (Solanum tuberosum L.) n class="Chemical">PONE-D-20-07306R3 Dear Dr. Han, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Mayank Gururani Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #3: (No Response) ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #3: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: Yes ********** 4. Have the authors made all data underlying the findinn class="Gene">gs inpan> their manpan>uscript fully available? The PLOS Data policy requires authors to make all data underlying the findinn class="Gene">gs described inpan> their manpan>uscript fully available without restriction, with rare exception (please refer to the Data Availability Statemenpan>t inpan> the manpan>uscript PDF file). The data should be provided as part of the manpan>uscript or its supportinpan>g inpan>formation, or deposited to a public repository. For example, inpan> addition to summary statistics, the data poinpan>ts behinpan>d meanpan>s, medianpan>s anpan>d varianpan>ce measures should be available. If there are restrictions on publicly sharinpan>g data—e.g. pan> class="Species">participant privacy or use of data from a third party—those must be specified. Reviewer #3: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #3: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #3: (No Response) ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #3: No 15 Oct 2020 n class="Chemical">PONE-D-20-07306R3 Transcriptome analysis reveals Nitrogen class="Disease">n deficiency inpan>duced alterations inpan> leaf anpan>d root of three cultivars of pan> class="Species">potato (Solanum tuberosum L.) Dear Dr. Han: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Mayank Gururani Academic Editor PLOS ONE
  18 in total

Review 1.  Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production?

Authors:  Allen G Good; Ashok K Shrawat; Douglas G Muench
Journal:  Trends Plant Sci       Date:  2004-12       Impact factor: 18.313

2.  Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary.

Authors:  Xizeng Mao; Tao Cai; John G Olyarchuk; Liping Wei
Journal:  Bioinformatics       Date:  2005-04-07       Impact factor: 6.937

3.  Nitrogen Deficiency Induced Alterations in the Root Proteome of a Pair of Potato (Solanum tuberosum L.) Varieties Contrasting for their Response to Low N.

Authors:  Anna Maria Jozefowicz; Anja Hartmann; Andrea Matros; Annegret Schum; Hans-Peter Mock
Journal:  Proteomics       Date:  2017-12       Impact factor: 3.984

4.  Genomic basis for coral resilience to climate change.

Authors:  Daniel J Barshis; Jason T Ladner; Thomas A Oliver; François O Seneca; Nikki Traylor-Knowles; Stephen R Palumbi
Journal:  Proc Natl Acad Sci U S A       Date:  2013-01-07       Impact factor: 11.205

5.  Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.

Authors:  Cole Trapnell; Adam Roberts; Loyal Goff; Geo Pertea; Daehwan Kim; David R Kelley; Harold Pimentel; Steven L Salzberg; John L Rinn; Lior Pachter
Journal:  Nat Protoc       Date:  2012-03-01       Impact factor: 13.491

6.  Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1.

Authors:  Zorica Kotur; Nenah Mackenzie; Sunita Ramesh; Stephen D Tyerman; Brent N Kaiser; Anthony D M Glass
Journal:  New Phytol       Date:  2012-03-20       Impact factor: 10.151

Review 7.  Understanding nitrate uptake, signaling and remobilisation for improving plant nitrogen use efficiency.

Authors:  Surya Kant
Journal:  Semin Cell Dev Biol       Date:  2017-08-30       Impact factor: 7.727

Review 8.  Cytosolic glutamine synthetase: a target for improvement of crop nitrogen use efficiency?

Authors:  Hanne C Thomsen; Dennis Eriksson; Inge S Møller; Jan K Schjoerring
Journal:  Trends Plant Sci       Date:  2014-07-10       Impact factor: 18.313

9.  Differential expression analysis for sequence count data.

Authors:  Simon Anders; Wolfgang Huber
Journal:  Genome Biol       Date:  2010-10-27       Impact factor: 13.583

10.  Transcriptome analysis of potato shoots, roots and stolons under nitrogen stress.

Authors:  Jagesh Kumar Tiwari; Tanuja Buckseth; Rasna Zinta; Aastha Saraswati; Rajesh Kumar Singh; Shashi Rawat; Vijay Kumar Dua; Swarup Kumar Chakrabarti
Journal:  Sci Rep       Date:  2020-01-24       Impact factor: 4.379
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  9 in total

1.  Comparison of single-trait and multi-trait genomic predictions on agronomic and disease resistance traits in spring wheat.

Authors:  Kassa Semagn; José Crossa; Jaime Cuevas; Muhammad Iqbal; Izabela Ciechanowska; Maria Antonia Henriquez; Harpinder Randhawa; Brian L Beres; Reem Aboukhaddour; Brent D McCallum; Anita L Brûlé-Babel; Amidou N'Diaye; Curtis Pozniak; Dean Spaner
Journal:  Theor Appl Genet       Date:  2022-06-23       Impact factor: 5.574

2.  Transcriptome analysis reveals multiple effects of nitrogen accumulation and metabolism in the roots, shoots, and leaves of potato (Solanum tuberosum L.).

Authors:  Heng Guo; Xiuqin Pu; Hao Jia; Yun Zhou; Guangji Ye; Yongzhi Yang; Tiancang Na; Jian Wang
Journal:  BMC Plant Biol       Date:  2022-06-09       Impact factor: 5.260

3.  Physical mapping of QTL associated with agronomic and end-use quality traits in spring wheat under conventional and organic management systems.

Authors:  Kassa Semagn; Muhammad Iqbal; Hua Chen; Enid Perez-Lara; Darcy H Bemister; Rongrong Xiang; Jun Zou; Muhammad Asif; Atif Kamran; Amidou N'Diaye; Harpinder Randhawa; Brian L Beres; Curtis Pozniak; Dean Spaner
Journal:  Theor Appl Genet       Date:  2021-07-31       Impact factor: 5.699

4.  Genome-based prediction of agronomic traits in spring wheat under conventional and organic management systems.

Authors:  Kassa Semagn; Muhammad Iqbal; José Crossa; Diego Jarquin; Reka Howard; Hua Chen; Darcy H Bemister; Brian L Beres; Harpinder Randhawa; Amidou N'Diaye; Curtis Pozniak; Dean Spaner
Journal:  Theor Appl Genet       Date:  2021-11-01       Impact factor: 5.699

Review 5.  Targeting Nitrogen Metabolism and Transport Processes to Improve Plant Nitrogen Use Efficiency.

Authors:  Samantha Vivia The; Rachel Snyder; Mechthild Tegeder
Journal:  Front Plant Sci       Date:  2021-03-01       Impact factor: 5.753

6.  Physical Mapping of QTL in Four Spring Wheat Populations under Conventional and Organic Management Systems. I. Earliness.

Authors:  Kassa Semagn; Muhammad Iqbal; Hua Chen; Enid Perez-Lara; Darcy H Bemister; Rongrong Xiang; Jun Zou; Muhammad Asif; Atif Kamran; Amidou N'Diaye; Harpinder Randhawa; Curtis Pozniak; Dean Spaner
Journal:  Plants (Basel)       Date:  2021-04-23

Review 7.  Germplasm, Breeding, and Genomics in Potato Improvement of Biotic and Abiotic Stresses Tolerance.

Authors:  Jagesh Kumar Tiwari; Tanuja Buckseth; Rasna Zinta; Nisha Bhatia; Dalamu Dalamu; Sharmistha Naik; Anuj K Poonia; Hemant B Kardile; Clarissa Challam; Rajesh K Singh; Satish K Luthra; Vinod Kumar; Manoj Kumar
Journal:  Front Plant Sci       Date:  2022-02-07       Impact factor: 5.753

8.  Identification of Spring Wheat with Superior Agronomic Performance under Contrasting Nitrogen Managements Using Linear Phenotypic Selection Indices.

Authors:  Muhammad Iqbal; Kassa Semagn; J Jesus Céron-Rojas; José Crossa; Diego Jarquin; Reka Howard; Brian L Beres; Klaus Strenzke; Izabela Ciechanowska; Dean Spaner
Journal:  Plants (Basel)       Date:  2022-07-20

9.  Screening of differentially expressed microRNAs and target genes in two potato varieties under nitrogen stress.

Authors:  Yue Lu; Jingying Zhang; Zhijun Han; Zhongcai Han; Shuang Li; Jiayue Zhang; Haoran Ma; Yuzhu Han
Journal:  BMC Plant Biol       Date:  2022-10-08       Impact factor: 5.260
  9 in total

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