Introduction

Nitrogen (N) is one of the essential mineral nutrients for plants and a large amount of N was added into the soil. However, only 30–50% of the applied fertilizer N is actually captured by crops, and the left part can subsequently leach and contaminate ground water1. Over-utilization of N fertilizer has resulted in secondary salinization in Chinese greenhouse. The excessively accumulated anion ion in soil of greenhouse is nitrate ()2,3. Excess nitrate inhibited the growth of vegetables4. Secondary salinization is thought to be one of the most important factors that seriously limit the sustainable development of protected agricultural production in China.

Hemoglobins (Hbs) are ubiquitous proteins found in all kingdoms of life5. In plants, three types of hemoglobins exist: symbiotic, non-symbiotic, and truncated hemoglobins. Non-symbiotic hemoglobins (nsHbs) can be further divided into class 1 (nsHb1, GLB1) and class 2 (nsHb2, GLB2) proteins that exhibit different oxygen-binding properties and gene expression patterns6,7. NsHb genes have been isolated from a number of both dicot and monocot species, such as, Arabidopsis, barley, casuarina, rice, trema, soybean, tomato8, oak9, and maize10. NsHb1 is involved in various biotic and abiotic stress responses. For example, nsHb1 genes have been reported to be up-regulated by hypoxic, osmotic, and high salt6,10,11, short-term waterlogging12, treatments with salicylic acid, methyl jasmonic acid, ethylene, and H2O213, deficiency of phosphorus, potassium, and iron8, as well as dual culture with specific microsymbionts14. NsHb1 genes were also strong induced by nitrate, nitrite, or nitric oxide (NO)15,16.

NO is an important signaling molecule involved in the physiological processes of plants17. Multiple physiological functions of NO in plants have been reported, including the stimulation of seed germination, suppression of floral transition, mediation of stomatal movement and modulation of plant growth, development, plant maturation and senescence, as well as abiotic and biotic stresses response18,19,20,21,22. There are several potential pathways for generating endogenous NO in plants. Among them, nitrate reductase (NR) and nitric oxide synthase (NOS) are two key enzymes for NO synthesis in plants23. NsHb have been shown to be involved in scavenging of NO molecules24. NsHb1 exhibit an extremely high affinity for NO25,26. Alfalfa (Medicago sativa) Mhb1 has a potential link to the NO signaling pathway27. Studies of transgenic Arabidopsis and alfalfa root cultures overexpressing nsHb1 genes suggest that nsHb1 may be involved in NO-mediated pathways of hypoxic or early-growth states28,29. NsHb was also shown to be involved in NO metabolism in barley, as ectopic expression reduced the amount of NO released during hypoxia24.

Spinach (Spinacia oleracea L.) is one of the most popular leafy vegetables grown in Chinese greenhouses, particularly in the Dianchi River division of Yunnan province. It is also an excellent source of dietary vitamins and minerals such as calcium and magnesium. In our previous work, a subtractive suppression hybridization (SSH) library of spinach roots under nitrate stress was constructed and several nsHb ESTs were found4. The function of SoHb in response to excess nitrate stress has not been investigated yet. Here, we report the molecular cloning and functional characterization of SoHb from spinach. Expression of the SoHb gene was analyzed by real-time quantitative RT-PCR (qRT-PCR) in spinach grown under nitrate or other abiotic stresses. Ectopic expression in Arabidopsis conferred decreased tolerance to nitrate and other abiotic stresses.

Results

Cloning and characterization of a spinach hemoglobin gene

Based on the suppression subtractive hybridization (SSH) assay in our previous study4, a 409 bp EST representing a putative hemoglobin gene was strongly induced under nitrate stress. By 5′RACE and 3′RACE cDNA cloning, a 124 and 265 bp cDNA was generated, respectively. By comparing and aligning the EST sequence of hemoglobin, the 5′RACE and 3′RACE products, the full-length cDNA of the SoHb gene was obtained, and it was subsequently confirmed by sequencing. The full-length cDNA of SoHb (GenBank Accession No. KC142173) was 667 bp and contained a single ORF of 504 bp, flanked by stretches of 51 and 112 bp at the 5′ and 3′ untranslated regions, respectively (Figure S1). The ORF encoded a predicted polypeptide of 167 amino acid residues, with a predicted molecular mass of 18.71 kDa and pI of 8.97.

Sequence alignment, using DNAman, of the predicted amino acid residues of SoHb with different members of the Hb family clearly established that SoHb had similarity with other Hb proteins. SoHb showed the highest identity with Beta vulgaris of 84.21%. SoHb showed identity with Gossypium hirsutum, Malus x domestica, Arabidopsis thaliana, Solanum lycopersicum of 75.00%, 73.05%, 71.86%, 66.47%, respectively (Fig. 1a). The SoHb deduced protein contained all conserved amino acid of plant Hbs. These include the CD1 phenylalanine, C2 proline and F8 proximal histidine residues needed for heme binding, and the E7 distal histidine which is involved in ligand binding30. A cysteine residue, found in most known plant nonsymbiotic Hbs, was also present in SoHb.

Figure 1: Analysis of the deduced amino acid sequence of SoHb.
figure 1

(a) Multiple sequence alignment of SoHb. Alignment of the predict SoHb protein with other Hb proteins from different plants. Conserved residues of heme- and ligand-binding (distal (E7) and proximal (F8) His residues, Phe (CD1), Pro (C2)) are underlined. Cys residues are also underlined. The protein sequences shown in the diagrams were listed in the GenBank database under the following accession numbers: Spinacia oleracea (Acc. No. KC142174); Beta vulgaris (Acc. No. KF54998); Arabidopsis thaliana (Acc. No. AK227823); Malus×domestica (Acc. No. AY224132); Gossypium hirsutum (Acc. No. AF329368); Solanum lycopersicum (Acc. No. NM001247569). (b) Phylogenetic analysis of SoHb together with the other plant nsHbs. The phylogenetic tree for Hb from different plant species were constructed on the basis of the multiple alignments of deduced amino acid sequences performed using ClustalW with default parameter settings and computed using the NJ method in the MEGA 6. The database accession numbers were indicated in parentheses after plant names.

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To clarify the relationships between SoHb with the other Hb proteins, the phylogenetic tree was generated. The result showed that SoHb fell into the class 1 family of non-symbiotic hemoglobin (Fig. 1b).

Expression of SoHb in spinach under nitrate and other abiotic stresses

Expression profiling of the SoHb was carried out by qRT-PCR in spinach grown in hydroponic cultures. Organ specific study showed that SoHb expressed in both root and shoot, and the expression in root was higher than in shoot (Fig. 2a). qRT-PCR was also conducted to examine the expression pattern of SoHb in spinach under 100 mM nitrate treatment for 0, 0.5, 3, and 6 h. The result showed that the SoHb expression level increased gradually from 0.5 to 6 h-treatment, and reached highest expression in 6 h. The expression increased 45.27-fold after 6 h-treatment (Fig. 2b).

Figure 2: Expression characteristics analysis of SoHb.
figure 2

(a) Organ-specific expression of SoHb by qRT-PCR. (b) Expression of SoHb under 100 mM nitrate treatment for 0, 0.5, 3 and 6 h. (c) Expression of SoHb under PEG, NaCl, SA and H2O2 stresses. (d) Expression of SoHb under NO donor, No scavenger and inhibitor treatment. (e) Expression of SoHb under nitrate stress supplemented with NO donor, No scavenger and inhibitor. (f) Western blot analysis of SoHb under nitrate stress supplemented with NO donor, No scavenger and inhibitor. Values are means ± S.E. based on three replicates.

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To examine whether SoHb was induced by other abiotic stresses, spinach root was treated with various inducers including PEG, NaCl, and global signaling molecules SA, and H2O2. As shown in Fig. 2c, when treated with PEG, the transcripts of SoHb decreased after 0.5-h treatment and then increased after 3, and 6-h treatment. When treated with NaCl, the expression levels first increased gradually and then decreased after 6-h treatment. The transcripts level of SoHb decreased gradually with the increasing H2O2 treatment time. After SA treatment, the SoHb expression reached highest expression in 0.5 h, then the expression decreased gradually.

Since Hb is known to be involved in NO detoxification, SNP (an NO donor), GSNO (an NO donor), cPTIO (an NO scavenger), tungstate (NR inhibitor), and L-NAME (NOS inhibitor) were applied to the solution. The transcript level of SoHb was strongly induced by 100 μM SNP and 100 μM GSNO. When plants were treated with 100 μM cPTIO, the transcript level of SoHb was repressed strongly. When treated with tungstate and L-NAME, the SoHb expression was decreased (Fig. 2d). The expression levels of SoHb gene and protein were analyzed by qRT-PCR and western blot after SNP, cPTIO, tungstate, and L-NAME were added into the nitrate solution (Fig. 2e,f). The result showed that after adding SNP and GSNO to the nitrate solution for 24 h, compared with the nitrate or SNP and GSNO treatment alone, the SoHb gene expression level were decreased. When tungstate, L-NAME, or cPTIO were added into the nitrate solution, the SoHb gene and protein level were also inhibited.

To understand the relationship between the existence of NO and the expression of SoHb, the accumulation of NO in root tissues were analysed by confocal laser microscope using NO sensitive dye, 3-amino, 4-aminomethyl-2′,7′-difluorescein, diacetate (DAF-FM DA) as a NO detector. As shown in Figure S2, the intensity of fluorescence was dramatically increased by the addition of SNP or nitrate. When SNP was added into the nitrate solution, the NO fluorescence was lower than the SNP and nitrate treatment alone, but was higher than the control. When the root were treated with tungstate, L-NAME, or cPTIO, the intensity of the fluorescence was decreased compared with the control, but was still detectable.

Overexpression of SoHb decreased the NO content in Arabidopsis

To investigate the biological function of SoHb, transgenic Arabidopsis plants were created under the control of the CaMV 35S promoter. The transgenic plants carrying SoHb were detected by PCR after the first screening with 30 mg/mL kanamycin. 20 kanamycin resistant putative transformants were obtained. Using specific primers, the SoHb gene was detected in the transgenic plants, while this gene was not detected in the WT plant (Figure S3a). The RT-PCR results showed that these kanamycin-resistant plants had significantly higher mRNA expression than the WT plants (Figure S3b). Western blot analysis revealed the presence of strong positive protein signals corresponding to SoHb in transgenic plants, while no signal was detected in WT plant (Figure S3c). Two lines (Hb-1, Hb-3) were selected for further analysis.

The NO contents of the two transgenic lines were assayed by the DAF-FM DA staining. The results indicated that two transgenic lines showed lower NO contents in root compared with the WT. When the Arabidopsis plants were treated with100 mM nitrate stress, the NO accumulation in all the tested lines were found, but the increase in transgenic lines was dramatically less than that of the WT, especially in the Hb-3 line (Fig. 3).

Figure 3: The NO fluorescence in SoHb transgenic Arabidopsis grown in MS medium with or without nitrate stress treatment.
figure 3

(a) Endogenous NO in Arabidopsis root was visualised using the DAF-FM DA with a confocal lasers scanning microscope system. (b)Average fluorescence intensity levels of root tips of WT and transgenic lines. The experiment was replicated three times with similar results. Bars = 100 μM.

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Previous work provided compelling evidence that GLB1 proteins were primarily involved in modulating NO concentrations31. We hypothesized that endogenous NO in transgenic plants under nitrate stress might be reduced by constant SoHb expression, which can act as an NO scavenger. To test the hypothesis, seedling growth assay was performed on MS medium supplemented with 0, 30, 50, and 150 μM SNP. The root growth of both WT and transgenic plants was inhibited in the medium supplemented with 30 to 150 μM SNP (Fig. 4). However, the inhibition in WT plants was more than that in the transgenic plants. The fresh weight and root length decreased less in transgenic plants, especially in the Hb-3 lines. The seedling growth assay was also performed on MS medium supplemented with 0, 30, 50, and 150 μM GSNO. The root length and fresh weight were also higher in SoHb transgenic plants than the WT plants (Figure S4).

Figure 4: Effect of excess SNP on the growth of SoHb transgenic Arabidopsis for 7 days.
figure 4

Seedling morphology (a), root length (b), fresh weight (c) of the WT and transgenic lines grown on MS supplemented with 0, 30, 50, and 150 μM SNP. Bars = 1 cm. (d) The NO content of WT and transgenic plants under excess NO stress treatment. WT was wild type Columbia Arabidopsis; Hb-1 and Hb-3 were two homozygous transgenic Arabidopsis lines. (e) Average fluorescence intensity levels of root tips of WT and transgenic lines grown under different concentration of SNP. The experiment was replicated three times with similar results. Bars = 100 μM.

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The NO contents were also analysed by confocal laser microscope. Under normal conditions, the NO florescence signals were weaker in transgenic root than the WT. The NO contents were increased in all the plants root with the increasing of SNP concentration. However, NO florescence signals of the transgenic plants were weaker than the WT plants, especially in the Hb-3 line root (Fig. 4d).

Overexpression of SoHb decreased the nitrate stress tolerance of Arabidopsis

To analyze the nitrate stress tolerance, the germinated transgenic plants were planted in the MS medium with 100 and 200 mM nitrate. As shown in Fig. 5, overexpression of SoHb resulted in reduction of nitrate stress tolerance. The transgenic plants have significantly shorter root and lower fresh weight than the WT plant under 100 mM nitrate treatment (P < 0.05).

Figure 5: Effect of excess nitrate stress on the growth of SoHb transgenic Arabidopsis.
figure 5

Seedling morphology (a), root length (b), fresh weight (c) of the WT and transgenic lines grown on MS medium supplemented with 100 and 200 mM nitrate for 7 days.

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To investigate if exogenous SNP can alleviate the inhibition of root growth of the nitrate-stressed Arabidopsis, 5 μM SNP was added into the MS medium supplemented with 100 mM nitrate. The result showed that the root length of transgenic plants were longer than the nitrate-stressed plants after adding SNP (Figure S5).

To further demonstrate the tolerance of nitrate stress, 2-week-old T3 seedlings were irrigated every day with nitrate solution for 15 d. The phenotypic effects of the salt treatment were shown in Fig. 6a, the transgenic plants showed more seriously chlorotic symptoms than the WT plants. To determine the effects of nitrate on photosynthetic efficiency, we compared the maximum photochemical efficiency of PSII in the dark-adapted state (Fv/Fm). There was no difference of Fv/Fm between WT and the transgenic lines under normal conditions. Excess nitrate resulted in significant decreases in Fv/Fm of the transgenic plants compared with the WT plants (Fig. 6b,c). The low value of Fv/Fm (0.44, 0.25) in Hb-1 and Hb-3 lines after treatment indicated that the transgenic plants suffered more severe damage than the WT plants (0.59).

Figure 6: Effects of nitrate stress on the tolerance of adult transgenic seedlings.
figure 6

(a) Morphology of adult plants of transgenic Arabidopsis after nitrate treatment. (b) Images of Fv/Fm (bottom); F0 (top) was used as the control. The pseudocolor code depicted at the bottom of the image ranges from 0 (black) to 1.0 (purple). (c) Average Fv/Fm values. (d) The lipid peroxidation analysis of WT and transgenic plants after nitrate stress treatment. Plants were treated with 100 mM nitrate solution for 15 days. The experiment was replicated three times with similar results. Means denoted by different letters show significant differences at P < 0.05 according to Duncan test.

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The MDA contents, as an indicator for the degree of oxidative stress, were analysed. The MDA contents after nitrate stress treatment were elevated remarkably in all tested lines. However, it was clear that MDA content of WT (1.54 nmol g−1 FW) was significantly lower than those of the Hb-1 and Hb-3 transgenic lines (2.15, 7.20 nmol g−1 FW) (Fig. 6d), indicating that overexpression of SoHb in the Arabidopsis plants confers decreased detoxification ability.

The physiological and molecular response of SoHb transgenic plants to nitrate stress

To elucidate the physiological mechanisms underlying the decreased stress tolerance, activities of SOD and CAT in the transgenic lines and WT were analyzed after the nitrate treatment. As shown in Fig. 7, before nitrate treatment, there was no significant difference of SOD and CAT activities between the transgenic and WT plants. After nitrate treatment, the activities of SOD, and CAT decreased in the transgenic and WT plants, especially in the transgenic lines. After nitrate treatment, the SOD activities of Hb-1, Hb-3 and WT decreased by 21.72%, 11.11%, 4.61%, respectively; and the CAT activities of Hb-1, Hb-3 and WT decreased by 66.07%, 44.50%, 26.30%, respectively.

Figure 7: The physiological and molecular response of SoHb transgenic plants to nitrate stress.
figure 7

(a–d) The antioxidant enzyme activities and relative transcript levels of SOD and CAT of WT and transgenic plants. (e–h) The expression of stress-induced genes of RD22, RD29A, DREB2A, and P5CS1 in WT and transgenic plants assayed by qRT-PCR. Plants were treated with 100 mM nitrate solution for 15 days. The experiment was replicated three times with similar results. Means denoted by different letters show significant differences at P < 0.05 according to Duncan test.

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To partly confirm the results of gene expression at transcriptional levels, the expression of MnSOD and CAT1 were analyzed by qRT-PCR assay. Before the nitrate stress, mRNA levels of MnSOD and CAT1 of the transgenic lines were significantly higher than that of the WT. After the nitrate treatment, the mRNA abundance of the WT plants displayed the higher transcription level of MnSOD, and CAT1, whereas their transcriptions were decreased in the two transgenic lines.

To elucidate the further role of SoHb in stress tolerance, we examined the effects of SoHb on the transcript levels of several stress inducible genes including RD22 (Responsive to Dehydration 22), RD29A, DREB2A (Dehydration-Responsive Element-Binding Factor 2A), P5CS1 (delta1-Pyrroline-5-Carboxylate Synthetase 1). Under normal conditions, the expression levels of RD22, RD29A, DREB2A, P5CS1 were strongly higher than that of the WT (P < 0.05). In the presence of nitrate stress treatments, the expression of all of these stress-responsive genes increased in the WT plants, but decreased in the two transgenic lines (Fig. 7).

Overexpression of SoHb decreased the NaCl and osmotic stress tolerance of Arabidopsis

To further analyze the other abiotic stresses tolerance of the transgenic plants, the germinated transgenic plants seeds were grown in the MS medium with 100 and 200 mM NaCl and mannitol, mimics the salt and osmotic stress. As shown in Fig. 8, overexpression of SoHb in transgenic plants resulted in reduction of the stress tolerance of NaCl and mannitol. After NaCl treatment, the fresh weight of Hb-1, Hb-3, and WT plants decreased by 57.01%, 69.10% and 48.73%, respectively; and the root length of Hb-1, Hb-3, and WT plants decreased by 86.72%, 93.81%, and 51.50%. After the osmotic stress, the fresh weight and root length of Hb-1, Hb-3, and WT plants decreased by 47.31%, 83.23%, 46.84%, and 62.22%, 79.21%, 44.45%, respectively, indicating that overexpression of SoHb decreased the salt and osmotic stress tolerance.

Figure 8: Effect of NaCl and osmotic stress on the growth of SoHb transgenic Arabidopsis for 7 days.
figure 8

Seedling morphology (a), root length (b), fresh weight (c) of the WT and transgenic lines grown on MS supplemented with 0, 100, and 200 mM NaCl and mannitol. WT was wild type Columbia Arabidopsis; Hb-1 and Hb-3 were two homozygous transgenic Arabidopsis lines. The experiment was replicated three times with similar results. Bars = 1 cm.

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Influence of SoHb overexpression on plant development

It has been documented that NO can repress floral transition in Arabidopsis32. Thus, the flowering time of WT and SoHb overexpression transgenic lines was investigated. Our results indicated a decrease in the rosette leaf number of SoHb transgenic plants, while the days to flowering were earlier than the WT plants (Fig. 9a). In 28 d, the bolting percentage of Hb-1, Hb-3, and WT plants were 13.52%, 69.21%, and 89.70%, respectively (P < 0.05). Four key flowering genes CO (CONSTANTS), SOC (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS), FLC (FLOWEREING LOCUS C), and GI (GIGANTEA) were assayed for their expression by semi-quantitative RT-PCR (Fig. 9d). The result showed that the SoHb transgenic plants up-regulated SOC and GI expression and down-regulated FLC expression, while the expression of CO was not significant affected, in accordance with their earlier flowering phenotype.

Figure 9: Overexpression SoHb transgenic plants showed earlier flower time phenotype.
figure 9

(a) SoHb transgenic lines flowered earlier. WT and SoHb transgenic lines were grown in soil under 16 h light/8 h dark cycles and were photographed after 28 d of growth. (b) Rosette leaf number of WT and transgenic SoHb lines during flowering (n > 30 plants). (c) The percentage of flower plants of WT and transgenic SoHb lines (n > 30 plants). (d) SOC, FLC, CO and GI expression in WT and transgenic SoHb lines assayed by semi-quantitative RT-PCR. All experiments were repeated at least three times. Means denoted by different letters show significant differences at P < 0.05 according to Duncan test.

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Besides, NO also affect the root growth and later root development. SoHb overexpressing transgenic plants have less lateral root than the WT plants. The average number of lateral root of the WT plants was 7.31, while the average lateral root number of the transgenic plants Hb-1 and Hb-3 were reduced to 5.02 and 4.05, respectively (Figure S6).

Discussion

Plant Hbs exist ubiquitously in the plant kingdom. In this study, we identified a gene encoding Hb in spinach root, which was designated as SoHb. The analysis of the SoHb expression pattern in various plant organs showed that the gene was strongly expressed in root as compared with the shoot, suggesting a more prominent role for SoHb in root. Hbs play important roles in various physiological processes in plants. A class 1 hemoglobin gene of Arabidopsis thaliana, AtHB1, was highly induced under hypoxia, by sucrose addition6 and nitrate addition33. GhHb1 expression was induced by SA, H2O2 and NO13. In this study, SoHb was induced by excess nitrate. PEG and NaCl also induced the expression of SoHb, suggesting that it may be involved in various defense pathways. Besides, the SoHb expression was induced in NO-promoting conditions, such as nitrate stress, by a NO donor (SNP), and the expression was repressed when the seedlings were treated with a NO scavenger, NR and NOS inhibitor. The transcripts of OsnsHB1 gene were also induced by NO in cultured rice cells16. These results suggested that SoHb expression corresponds to the cellular concentration of NO and might be involved in the modulation of NO levels in spinach.

NO is an important signaling molecule with diverse physiological functions in plants34,35,36, but its exact role in the response of plants to nutritional stress is still under evaluation. NO signaling is a key component of the root growth response to nitrate in Zea mays L37. Inhibition of root elongation in maize by high external nitrate is likely to result from a reduction of nitric oxide synthase-dependent endogenous NO levels in maize root apical cells38. NO is produced by nitrate reductase (NR) as an early response to nitrate supply and that the coordinated induction of nsHbs could finely regulate the NO steady-state39,40. SNP markedly enhanced endogenous NO levels in root apices grown in high nitrate, but they had no effect on endogenous NO levels in maize root apical cells grown in low-nitrate solution38. In our study, after 24-h nitrate stress or SNP treatment, the NO content increased in the spinach and the Arabidopsis roots, indicating that NO was involved in the excess nitrate stress response in spinach or Arabidopsis.

Transgenic maize cells with reduced levels of nsHb1 proteins produce more NO than WT cells25, whereas cultured alfalfa root cells with an increased level of barley nsHb1 accumulate less NO than the control cells29. Endogenous NO increased dramatically in the salt-treated WT plants but not in the TrHb transgenic plants41. GhHb1-transgenic Arabidopsis seedlings were more tolerant to exogenous NO and contained lower levels of cellular NO than the WT control13. In our experiment, the endogenous NO content in SoHb-overexpression plants was lower compared with the WT plants. Upon treatment with SNP, the seedling growth was less retarded in overexpressing SoHb transgenic lines compared with the WT seedlings.

Recent studies have reported the functional analysis of class 1 nsHb in transgenic plants. A transgenic Arabidopsis culture overexpressing Arabidopsis Hb showed resistance to hypoxia28. Ectopic expression of ZmHb in transgenic tobacco has enhanced tolerance to submergence, salinity and osmotic stresses10. Ectopic overproduction of GhHb1 in Arabidopsis led to constitutive expression of the defense genes PR-1 and PDF1.2, and conferred enhanced disease resistance to Pseudomonas syringae and tolerance to V. dahliae13. The Arabidopsis glb1 mutant line has increased resistance to pathogens, which was reduced in 35S-GLB1 compared to Col-042. In our study, under excess nitrate, NaCl, and osmotic stress, SoHb-overexpression plants grew worse than WT plants, suggesting that Arabidopsis plants with increased levels of SoHb were more sensitive to these abiotic stresses. This might because NO was not available in sufficient amounts to stimulate the salt tolerance signal transduction pathway. Removing NO produced an abnormal defense mechanism in the transgenic plants41.

Increased salt stress results in enhanced accumulation of reactive oxygen species (ROS) that are harmful for plant cells in high concentrations and subsequently led to lipid peroxidation. To avoid oxidative damage, plants are equipped with efficient antioxidant systems that can protect them from deleterious effects of ROS. Previous studies have shown that the activities of antioxidant enzymes, such as superoxide dismutase (SOD), and catalase (CAT) are directly correlated with stress tolerance in plants, and an augmented ability to scavenge ROS has been observed in plants that grow in sublethal levels of stress43. It has been reported that NO protects plant cells against oxidative stress by reducing ROS accumulation under salt stress44,45,46. Under stress conditions, NO regulates antioxidant enzymes at the level of activity and gene expression, which can cause either enhancement or reduction of the cellular redox status47. Rapid NO accumulation in response to strong stress stimuli was occasionally linked to inhibition of antioxidant enzymes and a subsequent rise in hydrogen peroxide levels47. In our study, the lipid peroxidation levels of the overexpression SoHb transgenic plants were higher than that of the WT plants, suggesting that the transgenic plants were damaged seriously by nitrate stress treatment. The activities and the transcription of MnSOD and CAT1 in SoHb-overexpression plants decreased more than that of WT plants after nitrate treatment, indicating that SoHb-overexpression plants decreased the ROS scavenging ability, which might be responsible for more serious oxidative stress damage.

High salinity usually induces the expression of stress-responsive genes. RD22, RD29A, and DREB2A are known to be involved in responses to drought and salt stress48,49,50. Proline accumulation is benifical for stress tolerance51, and P5CS1 encodes a key enzyme in the biosynthesis of proline52,53. In our study, overexpression of SoHb plants increased the expression of RD22, RD29A, DREB2A, P5CS1 under normal growth conditions. However, the expression of these genes decreased under nitrate stress, indicating that the differences in salt tolerance between the WT and transgenic plants might be partly due to the decreased expression of RD22, RD29A, DREB2A, P5CS1.

NO suppressed CO and GI gene expression and enhanced FLC expression, which indicated that NO regulates the photoperiod and autonomous pathways32. GI integrates cellular signals from light sensory transduction and the circadian clock and activates CO54. CO acts as a mediator between the circadian clock and the control of flowering55. SOC promotes flowering of Arabidopsis, and soc1 mutant is a suppressor of flowering56. FLC is a central floral repressor gene acting in the control of vernalization or autonomously induced flowering57. Lines with GLB1 silencing had a significant delay of bolting and after bolting, shoots reverted to the rosette vegetative phase by formation of aerial rosettes at lateral meristems. Lines with overexpression of GLB1 or GLB2 bolted earlier than wild type plants58. Here, the expression of these genes was also regulated in transgenic SoHb plants. 28-day-old SoHb overexpression plants had a higher expression of SOC and GI, while lower FLC expression level than WT plants, which flowered earlier, consistent with previous results showing that NO represses the photoperiod and autonomous floral pathways through FLC32, suggesting that non-symbiotic plant hemoglobin controls bolting by scavenging the floral transition signal molecule, NO.

Methods

Plant growth and stress treatments

Spinach seeds (Spinacia oleracea L. cv. Chaoji) were germinated on moisture filter paper in an incubator at 28 °C. After 15 days, batches of 20 seedlings were transferred to a plastic tank (40 × 30 × 12 cm) with 10 L of nutrient solution containing Ca (NO3)2 2.5 mM, KNO3 5 mM, KH2PO4 0.78 mM, MgSO4 2 mM, H3BO3 29.6 μM, MnSO4 10 μM, Fe-EDTA 50 μM, ZnSO4 1.0 μM, H2MoO4 0.05 μM, CuSO4 0.95 μM. pH of the nutrient solution was adjusted to 6.0–6.5 by addition of 98% (w/w) H2SO4. The experiment was carried out in a greenhouse of Kunming University of Science and Technology. The light period was about 12 h, and the air temperature was 20–28 °C during the day and 13–18 °C during the night.

For excess nitrate, NaCl, dehydration, hydrogen peroxide (H2O2) and salicylic acid (SA) treatments, 4-week-old seedlings were transferred into solutions containing either 100 mM nitrate (KNO3 and Ca(NO3)2 provide the same mol of ), 10% polyethylene glycol (PEG) 6000 (w/v), 150 mM NaCl, 1 mM H2O2, 1 mM SA for 0, 0.5, 3, and 6 h. Shoot and root of spinach seedlings were taken after different treatments, immediately frozen in liquid nitrogen and stored at −80 °C until use.

For NO treatment, 4-week-old spinach seedlings were treated with the NO donor, sodium nitroprusside (SNP, 100 μM) and S-nitrosoglutathione (GSNO, 100 μM), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl-3-oxide (cPTIO, 100 μΜ), the NR inhibitor tungstate (100 μΜ), or the NOS inhibitor Nx-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 μΜ) with or without nitrate for 24 h under the same conditions as described earlier.

RNA and DNA extraction

Total RNA was extracted with RNAiso reagent (Takara, Dalian, China) according to the instruction of the manufacturer. The quantity and quality of the isolated total RNA was examined by spectrophotometry and gel electrophoresis, respectively.

Genomic DNA was isolated from the shoot of wild type (WT) and transgenic Arabidopsis plants according to the modified CTAB method59.

Molecular cloning and sequence analysis of SoHb

According to the EST sequence of non-symbiotic hemoglobin of our previous SSH library, two different primers, Hb-R1 (5′-CAACGCGAATCTAGTCACCTC-3′) and Hb-R2 (5′-ACTTAACCACTAGAGCTTCCTGC-3′) were designed and the 5′ region was amplified according to the instruction manual (5′RACE System for Rapid Amplification of cDNA Ends, Version 2.0; Invitrogen, Carlsbad, CA).

According to the sequence of the internal conservative, and the 5′RACE product, two different primers, Hb-F1 (5′-AAAATGTCAACTGTTCTGCCTC-3′), Hb-F2 (5′-CATTTTGAGGTGACTAGATTCGC-3′) were designed. The 3′ region of the gene was amplified by a nested PCR. The first cycle was done with Hb-F1 and B26 (5′-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTTT-3′) under the following condition: template was firstly denatured at 94 °C for 5 min followed by 25 cycles of amplification (30 sec at 94 °C, 30 sec at 55 °C and 1 min at 72 °C) and by extension at 72 °C for 10 min. The amplified PCR product was diluted 10-fold and used as template in the second PCR using Hb-F2 and B26 under the following condition: 94 °C for 5 min, 30 cycles of 94 °C for 30 sec, 55 °C for 30 sec, 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR product was gel purified and cloned into the pMD18-T plasmid vector (TaKaRa, Dalian, China), and sequenced.

By comparing and aligning the EST sequence, the 5′ RACE and the 3′ RACE product, the full-length cDNA sequence was obtained. The open reading frame (ORF) sequence was amplified via PCR using a pair of primers HB-FB (5′-ggatcc ATGAGTCTCGAAAATGTCAAC-3′, BamH I site underlined) and HB-RX (5′-ctcgagATGAACTTCTAAAATTGTC-3′, Xhol I site underlined) following the condition: 94 °C for 5 min, 30 cycles of 94 °C for 30 sec, 55 °C for 30 sec, 72 °C for 1 min and a final extension at 72 °C for 10 min. The PCR product was gel purified and cloned into the pMD18-T plasmid vector (Takara, Dalian, China), and sequenced.

DNA sequence data was analyzed using the National Center of Biotechnology (NCBI) web site (http://www.ncbi.nlm.nih.gov). The BLAST program was used to search for sequence homology. The protein theoretical molecular weight and isoelectric point prediction were performed using DNAStar software. The alignment of the deduced protein sequences was computed using the DNAMan software employing standard parameters. The phylogenetic tree was computed using the NJ method in the MEGA 6.

Expression analysis by qRT-PCR and semi-quantitative RT-PCR

For qRT-PCR, reverse transcription of RNA was carried out according to the instruction of the SYBR® PrimeScript™ RT-PCR Kit II (Takara, Dalian, China). qRT-PCR was performed using the iCycler iQ Real-time PCR detection system (Bio-Rad, Hercules, CA). A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified using software provided with the iCycler iQ real-time PCR detection system. To minimize sample variations, mRNA expression of the target gene was normalized relative to the expression of the house-keeping gene of spinach 18SrRNA or Arabidopsis Ubq1. Three replicates were run for each sample. Primers used in qRT-PCR analyses were listed in Table S1.

Semi-quantitative RT-PCR was employed to analyze the transcript levels of SoHb in transgenic Arabidopsis plants and the expression of flower related genes. The reaction solutions and programs of RT-PCR were the same as those for SoHb isolation and thermal cycles (30). In addition, the cDNAs were amplified by ACTIN with the same procedure so as to confirm the loading of equal amounts of total RNA. RT-PCR experiments were repeated three times and the PCR products were detected by 1% agarose gel. Primers used in semi-quantitative RT-PCR analyses were listed in Table S2.

SDS-PAGE and immunological analysis

Soluble proteins of plants were extracted from frozen material in a 25 mM Tris-HCl buffer (pH 7.6) with 1 mM MgCl2, 1 mM EDTA and a cocktail of protease inhibitors (aprotinin 5 mg mL−1, leupeptin 2 mg mL−1, pepstatin 0.1 mg mL−1, PMSF 1 M, Na3VO4 1 mM, NaF 5 mM). After denaturisation, equal amounts of protein (30 mg) were separated on an SDS-polyacrylamide gel (12% (v/w) polyacrylamide). Proteins were then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA).

After incubation with a rabbit polyclonal anti-SoHb antibody (1:10,000), proteins were detected using a goat peroxidase-conjugated anti-rabbit antibody (1:4,000; Sigma) and visualized using ECL chemiluminescence (Bio-Rad, Hercules, CA). The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG (1:5,000) (Santa Cruz Biotechnology, Inc.).

Measuring of endogenous NO

Endogenous NO in spinach and Arabidopsis root after different treatment was visualised using the fluorescent probe, 4-amino, 5-aminomethyl-2’,7’-difluorescein diacetate (DAF-FM DA, Sigma-Aldrich, St. Louis, Mo, USA)60. Root tips of 4-week-old spinach or 2-week-old Arabidopsis were loaded with 10 μM DAF-FM DA in 20 mM HEPES buffer, pH 7.4, for 30 min, washed three-times in fresh buffer for 15 min. The root tips were then examined with a confocal lasers scanning microscope system (Nikon), using standard filters and collection modalities for DAF-FM DA green fluorescence (excitation 485 nm; emission 515 nm). Treatments were repeated 5 times. Signal intensities of green fluorescence in the images were quantified using Image J software (http://rsb.info.nih.gov/ij/).

Binary expression vector construction and Arabidopsis transformation

The coding sequence of SoHb was amplified with the primer pair Hb-FB and Hb-RX, and subcloned into gateway entry vector pENTR-2B (VIB, Gent, Belgium). The destination vector was pK7m34GW2–8m21GW3 (VIB, Gent, Belgium). The Gateway LR Clonase plus Enzyme Mix (Invitrogen, Carlsbad, CA) was used to perform the LR reactions according to the manufacturer’s instructions to obtain the pK2GW7-SoHb. The reaction mixture was incubated overnight at 25 °C and transformed into E. coli DH5a competent cells. The recombinant clones were selected on LB-medium plates with 50 μg mL−1 spectinomycin. The constructs were introduced into Agrobacterium tumefaciens strain EHA105 by electroporation and transformed into WT Arabidopsis (Columbia ecotype) by the floral-dip method61.

The T1 seeds were germinated on half-strength Murashige and Skoog (MS) agar plates containing kanamycin (30 mg L−1). The T2 seeds were germinated on half-strength MS agar plates containing kanamycin (50 mg L−1), and the resistant plants were transferred to nursery soil to obtain homozygous T3 seeds. Then the genomic DNA PCR, RT-PCR and western blot confirmed the presence and expression of SoHb in transgenic Arabidopsis. The T3 homozygous lines were used for further physiological analyses.

Stress tolerance assays for Arabidopsis

The WT and T3 progeny resulting from self-fertilization of Arabidopsis transgenic plants seeds were surface-sterilized and germinated in sterile plates containing MS salt, 3% (w/v) sucrose, and 0.7% (w/v) agar, pH 5.8. To determine the effect of nitrate stress, germinated seeds were moved to MS medium supplemented with 100 and 200 mM nitrate (normal concentration in the MS medium of 40 mM was used as a control). To determine the effect of NO tolerance of transgenic plants, 0, 30, 50, and 150 μM SNP or GSNO was added into the MS medium. To investigate if exogenous SNP could improve the nitrate stress tolerance of transgenic plants, 5μM SNP was added into the MS medium supplemented with 100 mM nitrate. The seeds were geminated in controlled environment chambers at an irradiance of 140 μmol photons m−2 s−1, 22 °C and 60% relative humidity in a photoperiod of 8 h/16 h light/dark regime. Plates were placed vertically on shelves to facilitate comparison of root growth.

For seedling treatment, 12 seedlings each of 2-week-old WT and two transgenic Arabidopsis lines were grown in the same pot (3 pots per experiments; 3 repeats) with regular watering every 2 days, followed by watering every 2 days with a 100 mM nitrate solution for 15 days. At the end of the treatment, photographs were taken and physiological parameters were analyzed.

Lipid peroxidation assays

Lipid peroxidation of WT and transgenic Arabidopsis seedling shoots was determined after 100 mM nitrate treatment for 15 d by estimating the formation of malondialdehyde (MDA), a product of lipid peroxidation, using the thiobarbituric acid reaction method62.

Antioxidant enzymes activities assays

The transgenic seedlings after 100 mM nitrate treatment for 15 d were used for the enzyme analysis. 0.2 g of shoot tissues was homogenized in 2 mL of 0.05 M sodium phosphate buffer (pH 7.8, containing 1.0 mM EDTA, and 2% (w/v) PVP). The homogenate was centrifuged at 10,000 × g for 20 min at 4 °C, and the supernatant was used for all enzyme activity assays. All steps in the preparation of each enzyme extract were carried out at 4 °C. All spectrophotometric analyses were conducted using UV-2450PC spectrophotometer (Shimadzu, Tokyo, Japan).

SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) spectrophotometrically at 560 nm62. The reaction mixture consisted of 0.3 mL each of 0.75 mM NBT, 130 mM methionine, 0.1 mM EDTA-Na2, 0.02 mM riboflavin, sterilized water, and 1 mL of 50 mM sodium phosphate buffer (pH 7.8). The reaction was started by adding 0.5 mL of enzyme extract and carried out for 20 min at 25 °C under a light intensity of 300 μmol m−2 s−1. One unit of enzyme activity was defined as the quantity of SOD required to produce 50% inhibition of reduction of NBT.

CAT activity was measured as the decline in absorbance at 240 nm due to the decline of extinction of H2O2. The reaction mixture containing 25 mM sodium phosphate buffer (pH 7.0), 10 mM H2O2 and 0.1 mL enzyme extract. The reaction was started by adding H2O263.

Chlorophyll fluorescence assays

Chlorophyll fluorescence was analyzed with PAM (Pulse-Amplitude-Modulation) Chlorophyll Fluorometer (Heinz-Walz-GmbH, Effeltrich, Germany). The measurements of the maximal quantum yield of PSII (Fv/Fm) were done according to Bai et al.64.

Analysis of flowering time

WT and transgenic SoHb lines were grown in soil under 16 h light/8 h dark cycles and photographed after 28 and 33 d of growth. Flowering time was scored as the number of rosette leaves and days to flowering at the stage when the first flower appeared in wild-type and transgenic SoHb lines.

Statistical analysis

Values presented were means ± one standard deviation (SD) of three replicates. Statistical analysis was performed by analysis of variance (ANOVA) using DPS software. Difference between treatments was separated by the least significant difference (LSD) test at a 0.05 probability level. For statistical tests, each transgenic line was only compared to WT.

Additional Information

How to cite this article: Bai, X. et al. Overexpression of spinach non-symbiotic hemoglobin in Arabidopsis resulted in decreased NO content and lowered nitrate and other abiotic stresses tolerance. Sci. Rep. 6, 26400; doi: 10.1038/srep26400 (2016).