Identification of nitric oxide (NO)-responsive genes under hypoxia in tomato (Solanum lycopersicum L.) root

Flooding periods, as one probable consequence of climate change, will lead more frequently to plant hypoxic stress. Hypoxia sensing and signaling in the root, as the first organ encountering low oxygen, is therefore crucial for plant survival under flooding. Nitric oxide has been shown to be one of the main players involved in hypoxia signaling through the regulation of ERFVII transcription factors stability. Using SNP as NO donor, we investigated the NO-responsive genes, which showed a significant response to hypoxia. We identified 395 genes being differentially regulated under both hypoxia and SNP-treatment. Among them, 251 genes showed up- or down-regulation under both conditions which were used for further biological analysis. Functional classification of these genes showed that they belong to different biological categories such as primary carbon and nitrogen metabolism (e.g. glycolysis, fermentation, protein and amino acid metabolism), nutrient and metabolites transport, redox homeostasis, hormone metabolism, regulation of transcription as well as response to biotic and abiotic stresses. Our data shed light on the NO-mediated gene expression modulation under hypoxia and provides potential targets playing a role in hypoxia tolerance. These genes are interesting candidates for further investigating their role in hypoxia signaling and survival.

Differentially expressed genes (DEGs) in response to hypoxia and SNP-treatment (functional classification of NO and hypoxia-responsive genes). The number of 1144 genes were differentially regulated (Padj < 0.05) in response to 48 h SNP-treatment (792 down-and 352 up-regulated genes). After comparison with 1421 differentially regulated genes (897 up-and 524 down-regulated) under 48 h hypoxia (Padj < 0.05) 53 , it was observed that 395 DEGs, were concertedly regulated under both, hypoxia and SNP-treatment (Fig. 2). Among above-mentioned genes, 144 genes showed the opposite-while 251 genes showed similar regulation changes (154 up-and 97 down-regulated) (Fig. 2). For further analysis, only those 251 genes with similar regulation changes under NO and hypoxia were chosen for downstream biological pathway analysis. The list of all common differentially regulated genes between hypoxia and SNP-treatment is provided in Supplementary Table S1.   (Fig. 3). It must be noted that GO terms refer to the proteins encoded by the genes and therefore in some cases, the word activity is used in GO term results. Functional annotation of the regulated genes showed that their encoded proteins are mainly involved in catalytic activity (GO:0003824) (> 61%), transporter activity (GO:0005215) (17%) and binding (GO:0005488) (14%). Biological processes such as metabolic process (GO:0008152) (40%), response to stimulus (GO:0050896) (22%) and cellular process (GO:0009987) (22%) showed the highest percentage of regulated genes. The cellular component categories with the highest percentage of regulated genes were cell (GO:0005623) (67%), organelle (GO:0043226) (14%) and cell junction (GO:0030054) (13%) (Fig. 3). For a more detailed analysis of the biological pathways, we used a plant based database, MapMan 54 . MapMan categories 54 are based on ITAG2.3 annotations (Supplementary Table S1).
Validation of differentially expressed genes in response to SNP-treatment using qPCR. To validate RNA-Seq data, qPCR was performed on 17 regulated genes in response to SNP-treatment (Supplementary Table S2). We observed similar gene regulation changes (SNP-treated/control) between RNA-Seq and qPCR (Fig. 4). These data confirm the validity of the RNA-Seq results used in this study. The confirmation of RNA-Seq data under hypoxia is provided elsewhere 53 . Hypoxia and SNP-associated phytohormone related genes. It was observed that 23 hypoxiainduced phytohormone related genes responded to SNP-treatment. These genes were related to different phytohormone categories such as abscisic acid (Solyc04g008960), auxin (ATB2, Solyc03g006490, IAA14, AILP1 and PIN2), brassinosteroid (Solyc11g006270), ethylene (Solyc12g006380, DLO1, two transcripts annotated as DLO2, DMR6, Solyc06g066830, Solyc03g116260, Solyc09g089680, Solyc06g073580 as well as two nitrilase encoded transcripts MES3 and Solyc09g011140), gibberellin (GASA5, two transcripts annotated as GASA6 and Solyc06g067860), and jasmonate (two transcripts annotated as LOX1) (Fig. 5).
Cell wall related genes regulated in response to hypoxia and SNP-treatment. Several cell wall related genes showed regulation changes in response to hypoxia and SNP-treatment. These transcripts were related to cell wall synthesis (CSLD3, UGT85A2, UGT71B1, UGT73C1 and UGT73B3), Cell wall modification (XTH8, XTH5 and XTH24, EXPA6 and EXPA3 ) and cell wall degradation (RD22) (Fig. 11).

Discussion
Tomato is a commercially important edible crop 10 . Improving tomato fruit size during the domestication process has been achieved by compensating its stress tolerance 55 . Former studies showed that SNP application on tomato root enhances salt stress tolerance, exhibited in improved growth and higher chlorophyll content. In the same study, SNP-treatment resulted in lower lipid oxidation, higher activity of antioxidant enzymes (SOD, APX, GR and POD) as well as an increase in ascorbate and proline content 10 . These data suggest that exogenous NO application via SNP-treatment is a reliable system for investigating the NO effect during stress response in the plant. Several former studies investigated gene expression regulation in response to exogenous NO application using different NO donors in various plant species such as Arabidopsis 11,18,56 , cotton 57 and birch 58 . However, our knowledge about the role of hypoxia-induced NO on gene expression modulation in tomato root is still scarce.
The current study represents the comparative transcriptome modulation of tomato (cv. Moneymaker) root in response to long-term (48 h) hypoxia 53 and SNP-application. Long-term hypoxia, but not SNP-treatment, resulted in significantly (P < 0.05) lower root fresh weight and chlorophyll content (SPAD values) (Fig. 1a,d).
Under hypoxia as well as under SNP-treatment, 395 genes (28% of hypoxia-regulated genes), were concertedly regulated. Among them, 251 genes, corresponding to. 64% of common regulated genes, were similarly   (Fig. 2). It was also noticeable that the number of common down-regulated genes (154) in response to hypoxia and SNP-treatment was higher than up-regulated genes (97).
To validate the RNA-Seq. data, the expression changes of 17 differentially regulated genes were confirmed using qPCR. A high level of consistency between the qPCR and RNA-Seq expression data indicates the reliability of transcriptome data (Fig. 4).
Phytohormone associated genes showed regulation changes response to hypoxia and SNP-treatment. Genes belonging to different phytohormonal categories such as auxin, ethylene, jasmonate and gibberellin revealed regulation changes in response to NO application and hypoxia (Fig. 5). NO modulates auxin effect on root architecture. Transcriptional regulation of auxin related genes in response to exogenous NO application has already been reported 59 . Among auxin related DEGs in response to hypoxia and SNP-treatment in our study, polar auxin transporter PIN2 showed the highest up-regulation (12-fold) under hypoxia (Fig. 5). In Arabidopsis, ERFVII mediated repression of PIN2, has been shown to be associated with root bending under hypoxia in soil grown roots 60 . Since in this study tomato were cultivated in a hydroponic system, hypoxia might not cause the same response as in soil grown roots. These data suggest that polar auxin transport seems to be involved in root growth response under hypoxia. However, species-specific response, effects of growth condition as well as NO-dependent regulation of PIN2 expression remain unclear.  www.nature.com/scientificreports/ IAA14 showed down-regulation in response to hypoxia and SNP-treatment (Fig. 5). IAA14 is a negative regulator of auxin response factors ARF7 and ARF19, which are involved in lateral root initiation via induction of multiple LBD/ASLs such as LBD16/ASL18 and LBD29/ASL16 61 . It has been shown that overexpression of IAA14 leads to inhibition of lateral root formation in Arabidopsis thaliana 62 . Moreover, this indicates that IAA14 negatively regulates lateral root formation. This is in accordance with its downregulation in response to hypoxia, when adventive root formation is beneficial for low oxygen tolerance. Down-regulation of IAA14 in response to SNP in this study indicates that its regulation might be NO dependent. Recently, it has been shown that ERF VII TFs are involved in the regulation of lateral root formation through repression of auxin-induced genes (LBD16, LBD18, and PUCHI and IAA19) 63 . To what extent this process is associated with NO signaling, requires further investigation.
Regulation changes in ethylene related genes were observed in the current study (Fig. 5). Ethylene, one of the key regulators of hypoxia response, is involved in aerenchyma formation under flooding stress and has been shown to have cross talk with NO 64 . Recently, it has been reported that early ethylene-induced PHYTOGLOBIN1 (PGB1) acts as NO scavenger leading to the ERFVII stability and adaption to upcoming hypoxia 65 . Moreover, investigation of ein2-1 nos1/noa1 double mutant in A. thaliana revealed cross talk between ethylene, through      66 . These data indicate the importance of NO signaling and ethylene response during hypoxia.
Defense related phytohormones such as JA and SA have been shown to have an interplay with NO. In the current study, two transcripts being annotated as LOX1 (LIPOXYGENASE 1) showed regulation changes (Fig. 5). Mutation of LOX1 gene in Arabidopsis has been shown to modify both signaling and redox related response under cadmium stress 67 . Former studies in A. thaliana confirmed the expression induction of AOS and LOX2 in response to SNP application 68 . It has been shown that NO increased the expression of several JA biosynthetic genes among them LOX3, encoding the enzyme involved in the conversion of linolenic acid to 13(s) hydro peroxy octadecatrieonic acid 69 . Our result is in accordance with the above-mentioned studies and suggests a possible role for hypoxia-induced NO on regulation of some of the JA biosynthesis genes.
NO has been shown to play a role in regulating GA biosynthesis and signal transduction 70 . Both antagonistic and synergic interaction between NO and GA has been reported 71 . Five GA related genes showed down-regulation in our study (Fig. 3), among them, GASA5 has been reported to negatively regulate GA-induced flowering and stem growth 72 . However, its role during hypoxia in the root and its interaction with NO has not yet been investigated. Our results imply a cross talk between NO, hypoxia and different phytohormone related genes.

NO responsive TFs play role in diverse physiological processes and stress response. Group VII
ERFs (ERFVII) has been shown to play a key role in hypoxia sensing and signaling via oxygen and NO dependent N-end rule pathway 50,73,74 . NO sensing has been shown to be executed via oxidation of Cysteine residue of ERFVII TFs, followed by arginylation and ubiquitination for proteasomal degradation (N-degron pathway) 48,75 . An ERFVII member, RAP2.2, was significantly up-regulated in response to hypoxia and SNP-treatment (Fig. 6). It has been reported that RAP2.2 overexpressing lines exhibited an improved hypoxia survival response while knockout lines showed weaker survival rate compared to the wild type. Moreover, RAP2.2 regulates the expres- www.nature.com/scientificreports/ sion of hypoxia-responsive genes which their encoded enzymes are involved in sugar metabolism as well as fermentation pathways 76 . These data indicate the significant role of RAP2.2 in root response to hypoxia. Moreover, among regulated genes were several transcripts (DLO1, DLO2 and DMR6) encoding proteins belonging to 2OG and Fe(II) dependent oxygenase superfamily. In a recent study in Arabidopsis, it was demonstrated that loss of function of PRT1, involved in N-degron pathways of ubiquitin-mediated proteolysis, improves the plant immune system. DMR6 and DLO1 proteins were accumulated in the former study indicating their importance in regulating the basal defense system 77 .
Several members of the NAC TF family such as ATAF2, NAC102 and NAC032 have been shown to be regulated by NO 78,79 . In accordance with the former studies, two transcripts (Solyc04g009440 and Solyc11g017470) annotated as ATAF2/NAC081, showed up-regulation in the current study in response to hypoxia and SNP-treatment (Fig. 6). NAC TFs are involved in plant development and response to different abiotic stresses 80 . The expression of ATAF2/NAC081 TF in maize has been shown to be positively regulated by ZmPTF1. ZmPTF1 is a member of the basic helix-loop-helix (bHLH) family involved in phosphate starvation and drought tolerance as well as root development in maize. ZmPTF1 binds to the G-box element within the promoter of several TFs such as ATAF2/NAC081 and NAC30 81 . Up-regulation of the above-mentioned genes has been reported to be involved in root development and stress response 81 . These data are in line with the up-regulation of ATAF2/NAC081 TF in our study indicating that it might be involved in root hypoxia response. However, whether NO is involved in ATAF2/NAC081 regulation requires further investigation.
NAC083, also known as VND-INTERACTING2 (VNI2), showed down-regulation in response to hypoxia and SNP-treatment in the current study. NAC083/VNI2 is an ABA responsive TF which has been shown to be involved in plant stress response. High salinity has been shown to increase the expression of NAC083/VNI2 in an ABA-dependent manner. Moreover, NAC083 negatively regulates stress-induced leaf senescence through regulation of COLD REGULATED (COR) and RESPONSIVE TO DEHYDRATION (RD) genes 82,83 . Two abiotic stress marker genes, COR15A/B and RD29A/B have been shown to be regulated by direct NAC083/VNI2 binding to their promoter. In the current study, only RD22 showed down-regulation which is in line with NAC083/ VNI2 down-regulation 83 . Our data suggest that the down-regulation of NAC083/VNI2 under hypoxia might be NO-dependent. However, how hypoxia and NO results in repression of NAC083/VNI2 in the root, requires more investigations and might provide more insight into the role of NAC083/VNI2 in the regulation of hypoxia response.
WRKY7 showed up-regulation in the current study (Fig. 6). Up-regulation of WRKY encoding transcription factors, such as WRKY22, in response to submergence, has been reported in Arabidopsis, resulting in induction of immunity related marker genes 84 . wrky22 mutant showed the lower expression level of defense related genes after submergence 84 . These data indicate that there is a link between submergence induced hypoxia and defense in plants. However, the underlying signaling network, particularly the role of NO, is not yet unraveled.
REIL1, a member of the C2H2 zinc finger family, showed the highest up-regulation in our study in response to hypoxia (17.3-fold) and SNP-treatment (12.1-fold) (Fig. 6). REIL1 and REIL2 have been shown to be involved in A. thaliana leaf growth in the cold but not in normal temperature 85 . REIL1 provides an interesting candidate for further investigating its role in response to NO and hypoxia tolerance.
Genes related with ROS metabolism were regulated in response to hypoxia and SNP-treatment. Genes encoding different categories of ROS associated proteins such as peroxidases, oxidases, nitrilases, glutathione S-transferases as well as cytochrome P450 showed up-and down-regulation in the current study (Fig. 8).
Accumulation of ROS and reactive nitrogen species (RNS) is associated with low oxygen stress 53,86,87 . NO and its derivatives such as peroxynitrite (OONO -), dinitrogen trioxide (N 2 O 3 ), and nitrous acid (HNO 2 ) have been reported to be involved in the modification of cellular redox statues 88,89 .
Among the genes, regulated under hypoxia and SNP-treatment, two Cytochrome P450 encoding genes (CYP72A14 and CYP707A3), showed the highest up-regulation, > 30 and > 6-fold, respectively, in response to hypoxia (Fig. 8). Cytochrome P450 (CYP450) can convert toxic metabolites (e.g. superoxide anion, hydrogen peroxide and hydroxyl radical) to H 2 O 2 to prevent harmful effects on the cell. Therefore, CYP450s are considered as markers for oxidative stress.
High level of ROS has to be scavenged by the cellular antioxidant system consisting of enzymatic antioxidants (e.g. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidases (GPXs), thioredoxin (Trx)) and non-enzymatic antioxidants (e.g. ascorbic acid, glutathione (GSH), carotenoids) 90 . Accordantly, up-regulation of genes encoding different classes of ROS scavenging enzymes were observed. Among six regulated GST encoding genes in our study, GSTU8 showed the highest up-regulation (ca. 6-fold) under hypoxia (Fig. 8). It has been shown that Arabidopsis gstu8 mutant line does not demonstrate any phenotypic changes nor modifications in the glutathione profile. However, it became evident that interaction between different dehydroascorbate reductases (DHARs) mediates the link between ascorbate and glutathione pools to ensure glutathione associated signaling under excessive H 2 O 2 91 . MT2B, encoding metallothionein-like protein 2B, a ROS scavenger, showed down-regulation in our study (Fig. 12). It has been shown that MT2B down-regulation is associated with ROS accumulation and subsequent aerenchyma formation in rice 64 . This indicates that MT2B might be involved in hypoxia tolerance in tomato root. However, the role of NO during this process has not yet been addressed.
Hypoxia and SNP-treatment led to the down-regulation of AQP encoding genes. Members of two families of aquaporin (AQP) encoding genes (PIPs: 12 genes and TIPs: 7 genes) were down-regulated in our study in response to hypoxia and SNP-treatment (Fig. 9).
Scientific RepoRtS | (2020) 10:16509 | https://doi.org/10.1038/s41598-020-73613-z www.nature.com/scientificreports/ AQPs are involved in the transport of water and other small molecules such as ammonia, boron, CO 2 , H 2 O 2 and urea across membranes 92 . Moreover, AQPs have been shown to be involved in plant biotic and abiotic stress response 93 . Expression reduction of NtAQP1, a member of the PIP1 family, in tobacco, led to a decrease in hydraulic conductivity of the root and eventually reduced drought stress resistance 94 .
In accordance with our results, microarray analysis showed that O 2 deficiency resulted in the down-regulation of AQPs in Arabidopsis (Liu et al., 2005) and Avocado 95 . It has been shown that beside modifications in cytosolic Ca 2+ and H 2 O 2 level, low cytosolic pH during anoxia results in inhibition of hydraulic conductivity through a mechanism of pH dependent AQP gating 96 . During flooding, AQPs remain phosphorylated but closed due to the protonation of His193 in PIP2;1 of spinach plants 97 . Tobacco PIP1;3 has been shown to be potentially involved in the O 2 transmembrane transport 98 . Hypoxia stress in hydroponically grown tobacco, resulted in the up-regulation of PIP1;3 in the whole root 98 and its down-regulation in lateral (LR) but not adventitious (AR) roots. The latter study demonstrated that beside PIP1;3, PIP1;1 was also down-regulated after 2 days of hypoxia treatment. Moreover, down-regulation of other AQP encoding genes such as PIP1;2, PIP1;4, PIP2;1 in LR, was observed after one week of hypoxia treatment 99 . The authors did not observe any difference in hydraulic conductance (K r ) between hypoxic and aerated plants, indicating the efficiency of AR in root water transport in tobacco. The down-regulation of PIP genes in our study is in accordance with AR response in tobacco. However, our study was focused on the whole root (Fig. 9). It has been reported that hypoxia is associated with a lower Kr in some species but an unchanged Kr in the others 100,101 . Tomato plants grown in soil, showed an early negative root hydraulic conductivity in response to flooding without affecting stomata closure 102 . It remains to determine whether expression changes in PIPs encoding genes is correlated with the functionality of PIPs and Kr in tomato root in response to hypoxia.
TIPs are involved in water transport between the vacuole and cytoplasm and therefore play a role in the regulation of cellular turgor pressure in plants 93,103 . One of the down-regulated TIP encoding genes in our study was TIP2;2 ( Fig. 9). Overexpression of TIP2;2, in tomato, improved drought stress tolerance of transgenic plants through regulation of transpiration rate. This indicates that the water permeability rate across tonoplast is involved in drought stress tolerance 104 . Our data suggest that studying expression changes as well as activity of TIP2;2 during flooding induced hypoxia gives more insight into the importance of vacuole-cytoplasm water relation in flooding stress tolerance. It is noteworthy that protein storage vacuoles (PSVs) in stem cell niche and lytic vacuoles (LVs) in mature cell, contain distinct TIPs proteins in their membran 105 . Further studies are required to unravel the response of cell type and vacuole specific TIPs to nitric oxide and flooding.
NO transport through AQPs, as well as its potential role in expression regulation of AQPs, is not yet clear. In a human cell line expressing AQP1, NO permeability across the cell membrane was correlated with water permeability 106 . In the latter study, NO transport was significantly reduced after the addition of HgCl 2 , an aquaporin inhibitor. The authors concluded that NO transport by AQP1 controls intracellular NO levels and its consequences 106 . However, whether NO transport and controlling its cellular level during hypoxia can be executed via aquaporins in plant cells, needs further investigations.
This result indicates that little is known about the role of AQPs in response to hypoxia and the role of NO in this process is still unknown. Gene regulation does not necessarily correlate with protein amount and function. Transcriptional, post-transcriptional and posttranslational regulation can eventually affect the amount and the activity of AQPs. Moreover, the clear link between NO and AQPs under hypoxic conditions is not yet fully known. Our result indicates the necessity of future investigations to unravel the functional significance of AQPs and NO in root hypoxia tolerance.
Stress associated genes showed expression changes under hypoxia and SNP-treatment. Among stress related genes, SRO5 (SIMILAR TO RCD ONE 5) showed the highest up-regulation level in response to hypoxia (22.2-fold) in our study (Fig. 10). SRO5 has already been reported to be a common hypoxia-responsive gene, particularly in the root, throughout the plant kingdom 65,107,108 . The role of SRO5 in salt stress has been addressed in Arabidopsis. SRO5 overexpression led to a reduction in root H 2 O 2 content in response to salt stress, compared to WT and sro5 plants 109 . Our data suggest that SRO5 might play a role in alleviating H 2 O 2 level during hypoxia stress. Further investigations are required to unravel the link between hypoxia-induced NO and SRO5 in tomato root.
Among the stress related genes, Heat shock protein 23.6 (HSP23.6) showed the highest up-regulation under hypoxia. SNP application in Arabidopsis has shown that different members of the heat shock TF family are responsive to NO 110 . Moreover, HSPs and HSFs are also responsive to anoxia 111,112 . These data indicate that hypoxia-induced NO production might be involved in the regulation of HSPs. The possible cross talk between hypoxia and NO-mediated thermo-tolerance and HSPs chaperon function remains to be determined.
Comparison to former hypoxia and NO studies. Common regulated genes in response to hypoxia and SNP-treatment (FDR < 0.05) in our study were compared with the data obtained from a well-designed former study on Arabidopsis seedlings 48 . In the former study, 357 genes showed regulation changes in response to hypoxia in WT. Out of the above-mentioned genes, only four genes were shared with our study (Supplementary  Table S3). This might be related to the fact that in our study only roots were exposed to hypoxia and SNP in a hydroponic system but in the other study, the whole seedlings were under submergence. Moreover, the plant age, 5 weeks old tomato vs. Arabidopsis seedlings, might partially explain the low overlap of the regulated genes between both studies. Moreover, regulated genes in our study were compared with the genes regulated in a triple NO mutant (nia1nia2noa1-2)under normoxia relative to WT 48 . 70 out of 251 (28%) genes in the current study were also regulated in the triple NO mutant under normoxia. Concomitantly, 51 out of 70 genes (73%) were regulated similarly in both studies (Supplementary Table S4).
Scientific RepoRtS | (2020) 10:16509 | https://doi.org/10.1038/s41598-020-73613-z www.nature.com/scientificreports/ The Arabidopsis N-degron pathway mutants (prt6 and ate1ate2) have been reported to exhibit constitutive expression of several core hypoxia genes (e.g. ADH1, SUS4 and PDC1) under normoxia leading to better resistance against hypoxia 48 . To identify the possible targets of the N-degron pathway in our study, regulated tomato genes were compared with the above-mentioned mutants in Arabidopsis. The result showed 13 common genes between tomato and Arabidopsis prt6 mutant. 10 genes, among them SUS4, were up-regulated (Supplementary Table S5). Comparison of our results with Arabidopsis ate1ate2 mutant revealed that 12 genes were common between two data sets, with 8 genes showing similar regulation changes (Supplementary Table S6).
In summary, our data suggest an overlap in gene expression response to long-term hypoxia and SNP-treatment. The concertedly regulated genes belong to different biological categories such as phytohormone signalling and transcription factor related genes as well as genes which their encoded proteins are involved in various metabolic pathways such as redox regulation, transport across membrane, glycolysis and fermentation. It can be proposed that the identified genes in our study could be considered as targets of hypoxia-induced NO and requires more investigation to unravel their role in the anatomic and metabolic adjustment of long-term hypoxia response and tolerance in tomato root. Beside TFs and phytohormones, the emphasis of the future studies needs to be placed on investigating the function of redox regulated proteins and their interaction with NO under hypoxia. These findings are essential to understand the cellular control of stress induced ROS/RNS as signal molecules as well as harmful radicals for the cell during hypoxia. A schematic model is illustrated to summarize the genes addressed in the discussion section (Fig. 13). It is noteworthy that this study was conducted on the RNA extracted from the whole root. Future investigations on phenotyping with cell type map and Single-cell RNA-seq approach can provide a more precise view on the cell type specific response and gene expression changes in response to nitric oxide and flooding stress.

Methods
Plant material and growth conditions. Plant age and growth condition was the same for both hypoxia and SNP studies. The hypoxia treatment has been previously described in details 53 . Tomato plants (Solanum lycopersicum L. cv. Moneymaker) were grown on sand in the greenhouse (500 μmol photons/m 2 /s and 25 °C under a 14/10-h light/dark) for three weeks. During this time, a modified Hoagland nutrient solution containing 5 mM nitrate (NO 3 -), as described previously 113 , was used for treatment. Three-week-old plants were transferred to hydroponic plastic boxes containing 6 L of nutrient solution (pH 5.5). Roots were submerged in the nutrient solution and aerated by mild bubbling using aquarist air pumps (Hailea ACO-9620, Raoping, Guangdong, www.nature.com/scientificreports/ China) and air outlets (Tetratec, Osnabrück, Germany). The hydroponic boxes were covered with dark covers, so that the roots were kept in dark. Despite dark, the low pH of the nutrient solution around the root will facilitate the release of NO from SNP 114 . The hypoxia treatment was conducted on five weeks old plant roots using N 2 gas (≥ 99.99 Vol. %) (Air Liquide, Germany) for 48 h 53 . For SNP-treatment, the root of five weeks old plants was treated with final concentration of 500 μM SNP. The roots were harvested 48 h after the initiation of SNPtreatment in the solution for this study. Hypoxia was induced via root exposure to N 2 gas 53 . Both treatments were started at 8:00 a.m. and continued 48 h until the harvest time at 8:00 a.m. A Konica Minolta SPAD-502 chlorophyll meter was used to measure the relative chlorophyll levels of leaf #3 (the third leaf above cotyledones) 53 .
Read mapping and identification of differentially expressed genes. All the processes involved in data mapping and analysis has been described in a former study 53 . Adaptor clipped reads obtained from the NextSeq500 Illumina platform (LGC Biosearch Technologies, Berlin, Germany) were used for the following processes. After omitting short fragments and low quality reads, rRNA sequences were filtered. Remaining sequences were mapped to tomato reference genome (ITAG 2.4) (The Tomato Genome Consortium, 2012) using CLC Genomics Workbench (Qiagen, V. 7.5.5). Adaptor clipped reads obtained from the NextSeq500 Illumina platform (LGC Biosearch Technologies, Berlin, Germany. Sequencing data are deposited in the Sequence Read Archive (SRA) database (bioproject accession PRJNA553994) at the National Centre for Biotechnology Information (NCBI). The bioproject's metadata are available at https ://datav iew.ncbi.nlm.nih.gov/objec t/PRJNA 55399 4 reviewer = 684sto9a948tin240f0tlt1o1h.
The TMM (trimmed means of M values) 115 and the edgeR algorithm 116 were used for normalization and estimation of P-values, respectively. The algorithm edgeR was used for log fold change values. The P-values were adjusted for multiple testing 117 . All calculations were performed with the CLC Genomics Workbench software (Qiagen, V. 7.5.5). Differentially expressed genes (DEGs) (P adj < 0.05) (Supplementary Table S1) were selected for subsequent analysis. The FDR threshold was used for the P-value in multiple tests (P adj ). GO term enrichment analysis (P adj < 0.05), was performed using the Panther database 118,119 . For biological pathway analysis of differentially regulated genes, MapMan categories based on ITAG 2.3 annotations 54 were used. Heat maps of differentially regulated genes were created using MultiExperiment Viewer (MeV 4.9.0) 120 .
RNA isolation and cDNA synthesis. The whole root was snap-frozen and grounded in liquid N 2 . 250 mg of the homogenized grounded powder was used for total RNA extraction following phenol-chloroform extraction method 121 . The integrity of RNA was checked on 1.2% agarose gel. RNA concentration was quantified photometrically using NanoDrop (ND-1000, Thermo Scientific, Wilmington, DE, USA).
cDNA synthesis was performed with two μg DNase I-digested total RNA as template and using oligo-(dT) 18 and RevertAid H Minus First Strand kit (Thermo Scientific, Waltham, USA). qPCR primer design and assay. Primers for quantitative real-time PCR (qPCR) were calculated by QuantPrime 122 (Supplementary Table S2). qPCR reactions were performed in total volume of 5 μl including 2.5 μl Power SYBR Green Master Mix (Thermo Fisher Scientific), 0.5 μM forward and reverse primers and 0.5 μl cDNA. ACTIN was used as reference gene 123 . The thermal profile used for all qPCRs was: 2 min 50 °C > 10 min 95 °C > (15 s 95 °C > 1 min 60 °C) 40x . Data were analyzed by the 2 −ΔΔCt method 124 .

Data availability
All materials and data sets represented in the current study are available in the main text or the supplementary materials. RNA-Seq. data are deposited to the Sequence Read Archive (SRA) database (bioproject accession PRJNA553994) at the National Center for Biotechnology Information (NCBI). The bioproject's metadata are available at https ://datav iew.ncbi.nlm.nih.gov/objec t/PRJNA 55399 4 reviewer = 684sto9a948tin240f0tlt1o1h.